Preparation and use of ginsentides and ginsentide-like peptides

ABSTRACT

The present invention relates to the methods of solid-phase peptide synthesis or recombinant production of ginsentide or ginsentide-like peptides or salts thereof. Further provided are uses of the ginsentide or ginsentide-like peptides or salts thereof as al-adrenergic receptor antagonists and vasorelaxants, nitric oxide-boosting agents, anti-thrombotic agents, anti-atherosclerotic agents, as protective agents against doxorubicin-induced cardiotoxicity, anti-ageing and adaptogenic agents, nutraceuticals, health supplements, or cosmetic ingredients.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. patent applicationSer. No. 16/348,770, filed May 9, 2019, which is a 371 U.S. NationalStage Application of International Application No. PCT/SG2017/050561,filed Nov. 9, 2017, which makes reference to and claims the benefit ofpriority of the Singapore Patent Application No. 10201609388X filed onNov. 9, 2016, the content of which is incorporated herein by referencefor all purposes, including an incorporation of any element or part ofthe description, claims or drawings not contained herein and referred toin Rule 20.5(a) of the PCT, pursuant to Rule 4.18 of the PCT.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 692148_401D1_SEQUENCE_LISTING.txt. The text fileis 327 KB, was created on Oct. 18, 2021, and is being submittedelectronically via EFS-Web.

FIELD OF THE INVENTION

The present invention relates generally to methods of solid-phasepeptide synthesis or recombinant production of ginsentide orginsentide-like peptides with native disulfide bonds or salts thereofand uses of the prepared ginsentide or ginsentide-like peptides or saltsthereof.

BACKGROUND OF THE INVENTION

Ginseng is a traditional herbal medicine that has been used forthousands of years. It is one of the most widely used and the mostvaluable of all medicinal plants. Ginseng has proven to be generallysafe based on its exceptionally long and widespread uses.

Plant peptides and proteins are usually underexplored as bioactiveconstituents for medicinal plants. Ginsentides are a class ofcysteine-rich peptides in ginseng that may be exploited as bioactivepeptides and drug discovery leads. However, isolation of ginsentidesfrom plants is usually laborious and costly. Therefore, there is need inthe art for methods of preparing ginsentides.

SUMMARY OF THE INVENTION

The present invention satisfies the aforementioned need in the art byproviding synthetical and recombinant methods of preparing ginsentidesor ginsentide-like peptides and uses thereof.

In a first aspect, the invention relates to a method of solid-phasepeptide synthesis of a ginsentide or ginsentide-like peptide or apharmaceutically acceptable salt thereof, the method comprising thesteps of:

-   -   (a) synthesizing the ginsentide or ginsentide-like peptide on a        solid support by stepwise coupling of Fmoc-protected, optionally        further suitably side-chain protected, amino acids, dipeptides        and/or oligopeptides in a linear C-terminal to N-terminal        fashion, wherein addition of the glycine residue at the position        corresponding to position 24 of SEQ ID NO:1 is mediated by        Fmoc-(Dmb)Gly-OH;    -   (b) cleaving the ginsentide or ginsentide-like peptide from the        solid support and deprotecting the peptide; and subsequently,    -   (c) performing organic oxidative folding of the ginsentide or        ginsentide-like peptide to form native disulfide connections.

In various embodiments, step (a) comprises the steps of:

-   -   (i) deprotecting a first amino acid linked to the solid support        by removing protective chemical groups from the first amino        acid;    -   (ii) activating chemical groups on a second amino acid to        prepare the second amino acid for coupling with the first amino        acid;    -   (iii) coupling the activated second amino acid to the        deprotected first amino acid to form a peptide from the first        and second amino acids; and    -   (iv) successively deprotecting, and coupling a plurality of        amino acids into the growing peptide chain until the ginsentide        or ginsentide-like peptide is synthesized.

In various embodiments, the method comprises accelerating at least oneof the deprotecting, activating, and coupling steps by applyingmicrowave energy.

In various embodiments, the organic oxidative folding is performed innon-aqueous conditions and performed in organic solvents.

In various embodiments, the organic oxidative folding comprises foldingthe ginsentide or ginsentide-like peptide in an organic solvent,preferably DMSO and/or isopropanol, comprising cysteamine and/ormorpholine.

In various embodiments, the organic oxidative folding is performed at 4°C.

In various embodiments, the method comprises purifying the foldedginsentide or ginsentide-like peptide or salt thereof, preferably byRP-HPLC.

In a second aspect, the invention relates to a method of recombinantlypreparing a ginsentide or ginsentide-like peptide or a pharmaceuticallyacceptable salt thereof, the method comprising the steps of:

-   -   (a) providing a host cell comprising a polynucleotide, wherein        the polynucleotide encodes a polypeptide comprising        Maltose-binding protein having the amino acid sequence set forth        in SEQ ID NO:65, a enterokinase cleavage sequence having the        amino acid sequence set forth in SEQ ID NO:66, and the        ginsentide or ginsentide-like peptide or salt thereof;    -   (b) culturing the host cell in a growth medium under conditions        allowing production of the polypeptide, and recovering the        polypeptide from the medium; and    -   (c) generating the ginsentide or ginsentide-like peptide or salt        thereof by cleaving the polypeptide using enterokinase.

In various embodiments, the polypeptide has the amino acid sequence setforth in any one of SEQ ID NOs: 67-80.

In various embodiments, the method further comprises purification of therecombinantly produced polypeptide prior to the enterokinase cleavage,preferably by amylose resin purification.

In various embodiments, the method further comprises purification of theginsentide or ginsentide-like peptide or salt thereof by anion-exchangechromatography and/or reverse-phase chromatography after theenterokinase cleavage.

In a third aspect, the invention relates to use of a ginsentide orginsentide-like peptide or a pharmaceutically acceptable salt thereof orenriched fractions by chromatographic, size-exclusion, or methodsrelated to purification from ginseng plants or plants containingginsentides or ginsentide-like peptides thereof as an al-adrenergicreceptor antagonist and vasorelaxant.

In a fourth aspect, the invention relates to use of a ginsentide orginsentide-like peptide or a pharmaceutically acceptable salt thereof orenriched fractions by chromatographic, size-exclusion, or methodsrelated to purification from ginseng plants or plants containingginsentides or ginsentide-like peptides thereof as a nitricoxide-boosting agent.

In a fifth aspect, the invention relates to use of a ginsentide orginsentide-like peptide or a pharmaceutically acceptable salt thereof orenriched fractions by chromatographic, size-exclusion, or methodsrelated to purification from ginseng plants or plants containingginsentides or ginsentide-like peptides thereof as an anti-thromboticagent.

In a sixth aspect, the invention relates to use of a ginsentide orginsentide-like peptide or a pharmaceutically acceptable salt thereof orenriched fractions by chromatographic, size-exclusion, or methodsrelated to purification from ginseng plants or plants containingginsentides or ginsentide-like peptides thereof as ananti-atherosclerotic agent.

In a seventh aspect, the invention relates to use of a ginsentide orginsentide-like peptide or a pharmaceutically acceptable salt thereof orenriched fractions by chromatographic, size-exclusion, or methodsrelated to purification from ginseng plants or plants containingginsentides or ginsentide-like peptides thereof as a protective agentagainst doxorubicin-induced cardiotoxicity.

In an eighth aspect, the invention relates to use of a ginsentide orginsentide-like peptide or a pharmaceutically acceptable salt thereof orenriched fractions by chromatographic, size-exclusion, or methodsrelated to purification from ginseng plants or plants containingginsentides or ginsentide-like peptides thereof as an anti-ageing andadaptogenic agent.

In a ninth aspect, the invention relates to use of a ginsentide orginsentide-like peptide or a pharmaceutically acceptable salt thereof orenriched fractions by chromatographic, size-exclusion, or methodsrelated to purification from ginseng plants or plants containingginsentides or ginsentide-like peptides thereof as a nutraceutical, ahealth supplement, or a cosmetic ingredient.

In accordance with all the afore-described aspects of the invention, theginsentide or ginsentide-like peptide has (i) the amino acid sequenceset forth in any one of SEQ ID NO:1-64; or (ii) the amino acid sequencesharing at least 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, or 74%,preferably at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, or 84%,even more preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, or 94%, most preferably at least 95%, 96%, 97%, 98%, or 99%sequence identity with the peptide of (i) over its entire sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood with reference to the detaileddescription when considered in conjunction with the non-limitingexamples and the accompanying drawings.

FIG. 1. Schematic diagram of the adaptogenic effects of ginsentides.TP1: SEQ ID NO:1.

FIG. 2. Schematic flow for the chemical synthesis of ginsentides.

FIG. 3. Oxidative folding strategies for synthetic ginsentide TP1.

FIG. 4. Comparison of natural and synthetic Ginsentide TP1 using UHPLCand MALDI-MS.

FIG. 5. Comparison of natural and synthetic Ginsentide TP1 using 1D and2D NMR.

FIG. 6. Functional comparisons of natural and synthetic Ginsentide TP1on nitric oxide production.

FIG. 7. Recombinant expression scheme for ginsentides. Peptide fragmentDDDDK: SEQ ID NO:66.

FIG. 8. IPTG induced MBP-TP1 fusion protein expression.

FIG. 9. Purification of MBP-TP1 fusion protein by amylose column. S-Celllysate; FT-Column flowthrough; W1-1^(st) wash; W2-2^(nd) Wash; E-Elutedproteins

FIG. 10. Removal of MBP tag by enterokinase.

FIG. 11. Mass spectrometry profile before and after enterokinasecleavage.

FIG. 12. RP-HPLC and MALDI-TOF MS profiles of native and reduced kB1.Panel A and B show the RP-HPLC profiles of native and reduced kB1 with“N” and “R” representing native and reduced respectively. Panel C and Dshow the MS profiles of the peaks N and R accordingly. Panel E and F arethe zoom-in profile of MS results in panel C and D, which demonstratethe difference between these two masses.

FIG. 13. RP-UPLC profiles of oxidative folding of kB1 showing the effectof different bases. A. Conditions contained different concentrations ofpyridine from 5-50%; B. Conditions contained different concentrations ofimidazole from 5-15%; C. Conditions contained different concentrationsof morpholine from 5-30%. All conditions contained 100 μM peptide, 100mM cysteamine and 10% (v/v) DMSO. The profiles show the reactionperformed for 1 h. “R” and “N” represent the reduced and native form ofkB1. “I” represents intermediate form of kB1.

FIG. 14. RP-UPLC profiles of oxidative folding of kB1 showing the effectof cysteamine and DMSO as redox agents. “R” and “N” represent thereduced and native forms of kB1 while “I” indicates the foldingintermediate.

FIG. 15. RP-HPLC profiles of oxidative folding of kB1 with differentthiols as reducing reagents. A. Reduced kB1; B. Conditions containing 10mM mercaptoacetic acid; C. Conditions containing 10 mM2-mercaptoethanol; D. Conditions containing 10 mM cysteamine. “R” and“N” represent the reduced and native forms of kB1 while I indicates thefolding intermediate. All conditions contained 100 μM peptide, 100 mMthiol, 10% (v/v) DMSO, 85% 2-propanol and 5% morpholine.

FIG. 16. ¹H NMR spectra comparison of the natural kB1 (red) and thesynthetic one (blue). Both peptides were dissolved in 90% H₂O/10% D₂O atpH 4.3. The spectra were obtained by a NMR spectrometer (600 MHz) at298K.

FIG. 17. Scheme for the purification of ginsentides from plantmaterials.

FIG. 18. Ginsentide TP1, TP3 and TP8 antagonized phenylephrine-inducedaortic ring contraction.

FIG. 19. Ginsentide TP1 does not antagonized KCI-induced contractedaortic ring.

FIG. 20. D2O exchange of Ginsentide TP1 by 1D NMR.

FIG. 21. Amino acid sequence, second and tertiary structures ofginsentides Primary and secondary structure (top left), the overall layof synthetic and natural TP1 (top right), the CPK model of thehydrophobic surface of TP1 (middle panels) and the 3D structure of TP5and TP8 as determined by NMR (bottom panel). TP1: SEQ ID NO:1.

FIG. 22. Mass spectrometry profiles of the aqueous extracts of roots of(A) Panax ginseng, (B) Panax quinquefolius and (C) Panax notoginsengusing MALDI-TOF MS.

FIG. 23. Mass spectrometry profiles of the aqueous extracts of (A)roots, (B) seeds, (C) leaves and (D) flowers of Panax ginseng usingMALDI-TOF MS.

FIG. 24. De novo sequencing of ginsentide TP1. Enzymatic digestion ofS-reduced peptides by chymotrypsin and trypsin generated one majorfragment each with m/z values of 2459 (SEQ ID NO:131) and 2831 (SEQ IDNO:132), respectively. The sequence of fragments were deduced using theb-ions and y-ions generated from MALDI-TOF MS/MS.

FIG. 25. (A) HPLC profile of the partially S-reduced and S-alkylatedginsentide TP1. Peaks 1, 2, 3, N, and RA contained the 3SS, 2SS, 1SS,native peptide, and fully S-NEM alkylated peptides, respectively. Aschematic representation of ginsentide TP1 disulfide mapping is alsoshown; (B) The putative unfolding pathway of ginsentide TP1 asdetermined by disulfide connectivity mapping. Under our experimentalconditions, the Cys I-IV bond was the first to be reduced to generatethe 3SS species, followed by the Cys V-VIII bond, generating the 2SSspecies, then the Cys III-VII bond generating the 1SS species, andlastly the Cys II-VI bond. TP1: SEQ ID NO:1.

FIG. 26. Ginsentide-encoding transcripts from Panax ginseng, Panaxquinquefolius and Panax notoginseng deduced from de novo assembly oftranscriptome data from NCBI database. The transcriptome data used arelisted as follow: Panax notoginseng flower (SRX378878), Panaxquinquefolius flower (SRX062267), Panax ginseng flower (SRX181263),Panax ginseng flower (SRX378873), Panax notoginseng leaf (SRX378880),Panax quinquefolius seed (SRX529365), Panax ginseng root (ERX137460).SPase: signal peptidase. The amino acid sequences are set forth in SEQID NOs: 133-146.

FIG. 27. shows that ginsentide TP1 increases cellular NO synthesis fromhuman endothelial cells (HUVEC-CS). Comparatively, ginsentide TP1 showsa three-fold increase in potency and magnitude NO release than bothginsenosides Rb1 and Rg1. Ginsentide TP1, TP3, TP8 and synthetic TP1shows comparable cellular NO release.

FIG. 28. Ginsentide TP1 activates endothelial nitric oxide synthase.Representative western blot analysis on the dose-dependent andtime-dependent effects of ginsentide TP1 on phosphorylated eNOS (p-eNOS)accumulations in HUVEC-CS cells. All results were expressed asmean±S.E.M. from three separate experiments. * P<0.05 compared toginsentide tP1 treated-group.

FIG. 29. Ginsentide TP1 induced nitric oxide formation involves PI3K/Aktsignaling. HUVEC-CS cells were exposed to ginsentide TP1 and (A) MK 2206(Akt inhibitor) or (B) LY294002 (PI3K inhibitor) for 1 h. IntracellularNO production was measured by DAF-2 DA staining and expressed asnormalized fluorescence intensity (C) Representative western blotanalysis of time-dependent effects of ginsentide TP1 on Aktphosphorylation in HUVEC-CS cells. All results were expressed asmean±S.E.M. from three separate experiments. * P<0.05 compared toginsentide TP1 treated-group.

FIG. 30. D₂O exchange of Ginsentide TP1 by 1D NMR.

FIG. 31. Ginsentide TP1 does not show (A) cytotoxic activities in Huh7cells and (B) hemolytic effects. Ginsentide TP1 does not induce (C)IL-6, (D) IL-8, (E) IL-10, and (F) TNF-α release from THP-1 cells. LPSwas used as positive control. All results were expressed as mean±S.E.M.(n=3). *P<0.05 compared to control group.

FIG. 32. Ginsentide TP1 antagonizes P2y12 activation. Ginsentide TP1,ginsenosides Rb1 and Rg1 were tested in antagonist mode with the P2y12Arrestin Biosensor Assay using 6.4 nM 2-methylthio-ADP to induce P2y12activation. P2y12 activation was measured by relative luminescence unitsand expressed as percentage of inhibition relative to blank.

FIG. 33. D₂O exchange of Ginsentide TP1 by 1D NMR.

FIG. 34. Ginsentide TP1 inhibits platelet aggregation in vivo. 10 mg/kgginsentide TP1 was injected to SD rat intravenously for 20 min.Platelet-rich plasma was isolated by centrifugation. Plateletaggregation was examined upon ADP or arachidonic acid stimulation.

FIG. 35. Pull down assay followed by LC-MS/MS and western blot analysisrevealed that ginsentide TP1 interacts with ESAM.

FIG. 36. Ginsentide TP1 inhibited basal monocyte adhesion toendothelium. Ginsentide TP1 was co-incubated with CFSE-labelled THP-1monocytic cell and HUVEC-CS endothelial monolayer for 1 h. Adhesioninhibitory peptides (GRGDSP) was used as a control.

FIG. 37. Heparin-binding properties of ginsentides using heparin HPLCaffinity column. Ginsentide TP1, TP3 and TP9 retented in a heparin HPLCaffinity column, but not TP2, TP5, TP8 and SIGGIR (negative control).

FIG. 38. (A) Modeling the interaction between ginsentide TP1 (SEQ IDNO:1) and heparin sulfate using the ClusPro Version 2.0 server. (B)Comparison of the synthetic peptide Ac-KSGGAW-NH₂ (SEQ ID NO:147) andits alanine-substituted analogues (Ac-ASGGAW-NH₂ (SEQ ID NO:148),Ac-KAGGAW-NH₂ (SEQ ID NO:149) and Ac-KSGGAA-NH₂ (SEQ ID NO:150) onheparin binding using heparin HPLC affinity column.

FIG. 39. Representative images of zebrafish embryos after DOX-treatmentwith or without ginsentide.

FIG. 40. Heartbeat of zebrafish embryos after ginsentides-treatment.

FIG. 41. Comparison of ventricular contractility after co-treatment ofDOX with ginsentide.

FIG. 42. Cell viability of H9c2 cells after treatment with ginsentidesor ginsenosides.

FIG. 43. Cell viability of H9c2 cells after co-treatment of DOX withginsentides or ginsenosides Rg1, Rb1 and Re.

FIG. 44. Effects of ginsentides on cell viability in DOX-treated breastcancer cell line (MDA-MB-231).

FIG. 45. Plasma concentration of ginsentides after intravenous injectionand oral administration in mice.

FIG. 46. Effect of ginsentide (TP1) upon HUVEC-CS cellular proteome.Cells were treated with ginsentide and whole cellular proteome wasstudied through TMT based quantitative proteomic approach. (a) TMT ratiodistribution of the invented dataset. (b) Differently expressed proteinsin HUVEC-CS cells during ginsentide treatment. (c). Functionsclassification of differentially regulated proteins upon ginsentidetreatment. (d) Relative protein levels of TERF2 after ginsentidetreatment.

FIG. 47. Effect of ginsentide (TP1) on hypoxic HUVEC-CS cellularproteome.

FIG. 48. Effects of ginsentide on leukocyte adhesion to hypoxicendothelial cells.

FIG. 49. Effects of ginsentide on hypoxia-induced adhesion moleculeexpressions in endothelial cells.

FIG. 50. Effects of ginsentide on the accumulation of unfolded proteinin hypoxic endothelial cells.

FIG. 51. Effects of ginsentide on the unfolded protein response pathwayin hypoxic endothelial cells.

FIG. 52. Effects of ginsentide on apoptosis pathway in endothelialcells.

FIG. 53. Effects of ginsentides on the survivability of hypoxicendothelial cells.

FIG. 54. Ginsentides reduced unfolded proteins in cardiomyocyte,vascular smooth muscle cells, and myocytes.

FIG. 55. Ginsentides reduced reactive oxygen species production inhypoxic endothelial cells.

FIG. 56. Ginsentides increased nitric oxide production in hypoxicendothelial cells.

FIG. 57. Ginsentide-like precursor sequences from NCBI and OneKptranscriptome database. The amino acid sequences are set forth in SEQ IDNOs:151-200.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description refers to, by way of illustration,specific details and embodiments in which the invention may bepracticed. These embodiments are described in sufficient detail toenable those skilled in the art to practice the invention. Otherembodiments may be utilized and structural, and logical changes may bemade without departing from the scope of the invention. The variousembodiments are not necessarily mutually exclusive, as some embodimentscan be combined with one or more other embodiments to form newembodiments.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. The singular terms “a,” “an,” and “the” include pluralreferents unless context clearly indicates otherwise. Similarly, theword “or” is intended to include “and” unless the context clearlyindicates otherwise. The term “comprises” means “includes.” In case ofconflict, the present specification, including explanations of terms,will control.

In a first aspect, the invention relates to a method of solid-phasepeptide synthesis of a ginsentide or ginsentide-like peptide or apharmaceutically acceptable salt thereof, said ginsentide orginsentide-like peptide having (i) the amino acid sequence set forth inany one of SEQ ID NO:1-64; or (ii) the amino acid sequence sharing atleast 65%, preferably at least 75%, even more preferably at least 85%,most preferably at least 95% sequence identity with the peptide of (i)over its entire sequence, wherein the method comprises the steps of:

-   -   (a) synthesizing the ginsentide or ginsentide-like peptide on a        solid support by stepwise coupling of Fmoc-protected, optionally        further suitably side-chain protected, amino acids, dipeptides        and/or oligopeptides in a linear C-terminal to N-terminal        fashion, wherein addition of the glycine residue at the position        corresponding to position 24 of SEQ ID NO:1 is mediated by        Fmoc-(Dmb)Gly-OH;    -   (b) cleaving the ginsentide or ginsentide-like peptide from the        solid support and deprotecting the peptide; and subsequently,    -   (c) performing organic oxidative folding of the ginsentide or        ginsentide-like peptide to form native disulfide connections.

The terms “peptide”, “polypeptide” and “protein” are usedinterchangeably herein and refer to polymers of at least two amino acidsconnected by peptide bonds. The terms also encompass an amino acidpolymer that has been modified naturally or artificially; for example,by disulfide bond formation, glycosylation, lipidation, acetylation, orphosphorylation. The term “amino acid” refers to natural and/orunnatural or synthetic amino acids, including both the D and L opticalisomers, amino acid analogs (for example norleucine is an analog ofleucine) and derivatives known in the art. The term “natural aminoacid”, as used herein, relates to the 20 naturally occurring L-aminoacids, namely Gly (G), Ala (A), Val (V), Leu (L), Ile (I), Phe (F), Cys(C), Met (M), Pro (P), Thr (T), Ser (S), Glu (E), Gln (Q), Asp (D), Asn(N), His (H), Lys (K), Arg (R), Tyr (Y), and Trp (W). Generally, in thecontext of the present application, the peptides and polypeptides areshown in the N- to C-terminal orientation.

The term “pharmaceutically acceptable salt” as used herein means thosesalts of a peptide of interest that are safe and effective foradministration to a mammal and that possess the desired biologicalactivity.

The term “sequence identity” as used herein refers to the extent thattwo sequences are identical (i.e., on a nucleotide-by-nucleotide basisfor nucleic acids or amino acid-by-amino acid basis for peptides) over awindow of comparison. This is calculated by comparing two optimallyaligned sequences over the window of comparison, determining the numberof positions at which the identical nucleic acid base or amino acidresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the window of comparison (i.e., the window size), andmultiplying the result by 100 to yield the percentage of sequenceidentity.

It should be noted that the sequence identity is always calculated inrelation to a reference sequence over its entire length. In thisconnection, the term “window of comparison”, as used herein, refers to aconceptual segment of contiguous nucleotide or amino acid positionswherein a nucleotide or amino acid sequence may be compared to areference sequence and wherein the portion of the nucleotide or aminoacid sequence in the comparison window may comprise additions ordeletions (i.e., gaps) while the reference sequence does not compriseadditions or deletions for optimal alignment of the two sequences.

Optimal alignment of sequences for comparison may be conducted by thelocal homology algorithm of Smith and Waterman, Adv. Appl. Math. 1981,2:482; by the alignment algorithm of Needleman and Wunsch, J. Mol. Biol.1970, 48:443; by the search for similarity method of Pearson and Lipman,Proc. Nat. Acad. Sci. U.S.A. 1988, 85:2444; or by computerizedimplementations of these algorithms (including, but not limited toCLUSTAL in the PC/Gene program by Intelligentics, Mountain View, Calif.;and GAP, BESTFIT, BLAST, FASTA, or TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group (GCG), 575 Science Dr.,Madison, Wis., U.S.A.). The CLUSTAL program is well described by Higginsand Sharp, Gene 1988, 73:237-244; Higgins and Sharp, CABIOS 1989,5:151-153; Corpet et al., Nucleic Acids Res. 1988, 16:10881-10890; Huanget al, Computer Applications in the Biosciences 1992, 8:155-165; andPearson et al., Methods in Molecular Biology 1994, 24:307-331. Alignmentis also often performed by inspection and manual alignment.

It should be noted that the peptide sequences described herein onlydefine the primary structure of the peptides, i.e. the sequence of aminoacids, without including any information about the disulfide bridges(—S—S—) that may be existing in the peptides. Disulfide bridges areproduced by the oxidative folding of two different thiol groups (—SH)present in the peptides. The peptides described herein contain at leasteight different thiol groups (i.e. eight cysteine residues eachcontaining a thiol group) and may theoretically form zero, one, two,three, four, or more intramolecular disulfide bridges. However, itshould be understood that the peptides in the context of the wholeapplication comprise native disulfide connectivitiy (i.e. Cys I-IV, CysII-VI, Cys III-VII, and Cys V-VIII) as illustrated in Table 1.

Preferred embodiments of ginsentides and ginsentide-like peptides inaccordance with the present application are described in Table 1 andTable 2.

TABLE 2 Amino acid sequence of preferred ginsentide-like peptides SEQ IDSource Amino acid sequence 15 Angiopteris evectaCIPKGGWCLFDIMGCCKPCGCLAGFCVVVVGDDCN 16 Amaranthus retroflexusCVPKGTPCLYKPEPCCGANCFCDTSAYTFQYVCKCY 17 Agrostis stoloniferaCLPSGGFCMFRPTDCCGNCGCLYPVGVCYGSRCEE 18 Blasia sp.CLKNGEFCWGDPSGCCGNCGCLIIPGVCYGTGC 19 Bazzania trilobataCLNGGGYCGSFTREACCYNCVCMMAFCVCG 20 Coffea canephoraCSPFGKPCRYNPWGCCDSCVCVATPADEGRCLGNC 21 Daucus carotaCLPNGGFCMFRPMDCCGSCGCLYPVGVCFGTGC 22 Daucus carotaCYPKGHECRTDPTLCCHNCGCIMPVGVCFGINC 23 Eragrostis curvulaCLPSGGFCMFRPKDCCGSCGCLYPIGVCFGSSC 24 Eleusine coracanaCIPMGGFCLFNLRGCCGSCGCLAGFCVVRDASSCDL 25 Eleusine coracanaCIPMGGFCLGNLRGCCGSCGCLAGFCWRPASSCDS 26 Elymus wawawaiensisCLPSGGFCMFRPKDCCGNCGCLYPIGVCYGSRCEE 27 Gossypium hirsutumCKPKGSFCLFDLQSCCRPCGCLAGWCYNIDHDCNEYT 28 Griselinia littoralisCISSGGFCMFNPRDCCGSCGCLYPMGICYGSSC 29 Gossypium raimondiiCKPIGSFCLFDLTSCCRPCGCLAGFCYNLDHNCNEYT 30 Gossypium raimondiiCKPKGSFCLFDLTSCCRPCGCLAGWCYNYDHECNEYT 31 Gossypium raimondiiCIAKGGFCLFDLTSCCRPCGCLAGWCYNIDHDCKEYT 32 Gossypium raimondiiCIAKGGFCLFDLTSCCRPCGCLAGWCYNIDHDCNEYA 33 Gossypium raimondiiCIAKGGFCLFDLTSCCRPCGCLAGWCYNIDHDCNEYT 34 Hibiscus cannabinusCIPKGGWCLFDIMGCCKPCGCLAGFCVVVVGDDCN 35 Hibbertia grossulariifoliaCSPLGGKCGDLVECCSGCVCIVVPTYTCVGHC 36 Hedera helixCQPFDAPCDTFYGFYCCGSCTCTYVDFWHTSRCTGSC 37 Heracleum lanatumCLPAGGFCMFRPMDCCGTCGCLYPVGVCFGNDC 38 Lennoa madreporoidesCIGAGGFCMFNPMDCCGNCGCLYPVGICFGTGC 39 Microtea madreporoidesCLNGGGYCGSFTREACCYNCVCMMAFCVCG 40 Mollugo nudicaulisCKSAGEWCGFSVVTDCCNSCGCLAGFCYGTSC 41 Myodocarpus sp.CIPKGGFCLFDLRGCCGMCGCLAGVCFNYDHPCEE 42 Menyanthes trifoliataCLSSGGFCMFRPNDCCGNCGCLYPVGICYGTGC 43 Oryza sativaCLPAGGFCMFRPMDCCGNCGCLYPVGVCYGSRCEE 44 Pholisma arenariumCLPAGGFCMFRPMDCCGNCGCLYPVGVCYGSRCEE 45 Pholisma arenariumCISAGGFCFFDPMNCCGNCGCLYPVGICVGTNC 46 Polyscias fruticosaCSPLGGKCGDLVECCSGCVCIVVPTYTCVGHC 47 Polyscias fruticosaCIPLGGDCTDLFDCCPGCVCIITDLTCDGNCFRGA 48 Polyscias fruticosaCLTLGLYCGGGSGECCSGCLCVYPTLTCRGNCYRGA 49 Phyllanthus sp.CSDPGGYCVPFFQGCCNDCSCLDLGVVAGVCVCI 50 Populus trichocarpaCISSGGWCFTQPKNCCGNCGCLYPIGICFGSDC 51 Populus trichocarpaCISSGGWCFPNPKNCCGNCGCLYPIGICFGSDC 52 Populus tremulaCISSGGFCFTQPMNCCGNCGCLYPLGICYGSDC 53 Salix dasycladosCLPSGGFCMFQPMNCCGNCGCLYPIGVCYGSNC 54 Theobroma cacaoCLSAGGFCMFNPMDCCGNCGCLYPMGICYGSGC 55 Theobroma cacaoCLSAGGFCMFNPMDCCGNCGCLFPMGICYGSGC 56 Theobroma cacaoCLSAGGFCMFNPMDCCGNCGCLYPMGFCYGSGC 57 Theobroma cacaoCLSAGGFCMFNPMDCCGNCGCLYPLGFCYGSGC 58 Theobroma cacaoCLSAGGFCMFDPMDCCGNCGCLYPMGICYGSGC 59 Theobroma cacaoCLSAGGFCMFIPMDCCGNCGCLFLMGFCYGSGC 60 Theobroma cacaoCLSAGGFCMFIPMDCCGNCGCLFPMGFCYGSGC 61 Theobroma cacaoCLSAGGFCMFNPMDCCGNCGCLYPMGICYGSGC 62 Theobroma cacaoCPSAGGFCMFNPMDCCGNCGCLYPMGICYGSGC 63 Theobroma cacaoCLSAGGFCMFNPMDCCGNCGCLYPLGICYGSGC 64 Triticum aestivumCLPAGGFCMFRPMDCCGNCGCLYPAGVCYGTRCEE

Solid-phase peptide synthesis or ‘SPPS’ refers to the direct chemicalsynthesis of peptides, wherein an insoluble support is used as an anchorfor the growing peptide chain, which is typically built up one aminoacid at a time. The free N-terminal amine of a solid-phase attachedpeptide is coupled to an N-protected amino acid unit. This unit is thendeprotected, revealing a new N-terminal amine to which a further aminoacid unit may be attached. The general principle of SPPS is one ofrepeated cycles of such coupling-wash-deprotection-wash steps, adding,typically one amino acid at a time, until the peptide of the desiredsequence and length has been synthesized. As will be understood by thoseskilled in the art it is possible, in principle, to couple N-protectedpeptides instead of single amino acids to the growing chain in one ormore elongation cycles. The present invention also encompasses methodswherein one or more larger N-protected peptides, or oligopeptides,typically having a length of up to 20 amino acid, preferably up to 10amino acids, more preferably up to 5 amino acids, still more preferablyup to 4 amino acids are added to the growing chain. In a particularlypreferred embodiment, a method as defined herein is provided, whereinstep a) comprises stepwise coupling amino acids, dipeptides and/ortripeptides, preferably amino acids and/or dipeptides to the growingpeptide chain. In a most preferred embodiment of the invention, step a)comprises stepwise coupling of single amino acids or building blocks(e.g. amino acid pairs) to the growing peptide chain.

Preferably, in accordance with the present invention, the growingpeptide is anchored to the resin through the terminal carboxyl group.Nevertheless, the use of certain linkers allowing for anchoring of thegrowing peptide-chain via a side-chain residue, is also envisaged andmay even be preferred.

The solid support for SPPS typically is a solid, non-soluble supportmaterial. For the purposes of the present invention, such a solidsupport comprises sites for anchoring of a first amino acid (orpeptide). Such functional sites for anchoring of the peptide are termedlinkers. If needed, other linker moieties such as e.g. more specialized,for instance more acid-labile, linkers may be grafted to the first,integral linkers on the premade solid support, which is often thenreferred to as a ‘handle’. Polymeric organic resin supports are the mostcommon type of solid support material, typically comprising highlysolvated polymers with an equal distribution of functional groups.Examples include Polystyrene (PS); Polyacrylamide (PA); polyethyleneglycol (PEG); PEG-Polystyrene (PEG-PS) or PEG-Polyacrylamide (PEG-PA);and other PEG-based supports. The invention is not particularly limitedwith respect to the solid support material. The 2-chlorotritylchlorideresin, Wang resin (4-Benzyloxybenzyl Alcohol resin) and PAM resin(4-hydroxymethyl-phenylacetamidomethyl), are particularly suitable solidsupport materials for methods of the present invention. Other suitableexamples include, but are not limited to: PEG-HMPB (cross-linked PEGfunctionalized with 4-(4-Hydroxymethyl-3-methoxyphenoxy)butyric acid);Rink amide resin(4-(2′,4′-Dimethoxyphenyl-Fmoc-aminomethyl-phenoxy-resin); andMerrifield resin (copolymer of styrene and chloromethylstyrenecross-linked with divinylbenzene). Solid support materials should meetseveral requirements, besides being chemically inert and able towithstand the conditions of synthesis: solid support particles arepreferably of conventional and uniform size, mechanically robust, easilyfilterable and highly accessible to the solvents allowing thepenetration of the reagents and the enlargement of the peptide chainwithin its microstructure. Resins as used in the present invention aretypically of standard mesh size, which is about 50-500 mesh, morepreferably 100 to 400 mesh.

As stated above, the present method concerns so-called ‘Fmoc SPPS’methods, wherein Fmoc (Fluorenylmethyloxycarbonyl) N-protected aminoacids and peptides are added to the growing chain. Fmoc protection insolid support peptide synthesis has significant advantages because itsremoval involves very mild basic conditions (e.g. piperidine solution),such that it does not disturb the acid labile linker between the peptideand the resin. Fmoc N-protected amino acids are commercially available.Furthermore, reactions to produce Fmoc N-protected amino acids orpeptides are common general knowledge for those skilled in the art.

Each incoming amino acid that is added to the growing peptide chain ispreferably also protected, where suitable, with a side-chain protectinggroup, which is typically acid-labile. Protection groups suitable forthis purpose are well known in the art. Commonly employedcarboxy-protection groups for Glutamine and Aspartic acid are e.g. Mpe,O-1-Adamantyl, O-benzyl and even simply alkyl esters may be used, thoughless common. For the sake of ease, typically and preferably tert-butylgroups are used. Tyrosine may typically be protected by protectiongroups such as tert-butyl ether or Z- or more preferably2-Bromo-Z-esters. It is equally possible to use tritylalkohol protectiongroups such as 2-chloro-trityl or 4-methoxy or 4,4′ methoxy-tritylgroups. Preferably, a trityl or a tert-butyl (tBu) protection group isused, most preferably a tBu protection group, meaning the tyrosyl sidechain is modified to a tertiary-butyl ether. The tBu group is onlyefficiently removed under strongly acidic condition. Suitable Arginineprotective groups include2,2,4,6,7-pentamethyldihydrobenzofuranyl-5-sulfonyl (Pbf),adamantyloxy-carbonyl and isobornyl-oxy-carbonyl,2,2,5,7,8-pentamethylenchromanesulfonyl-6-sulfonyl (Pmc),4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr) and its4-tert.butyl-2,3,5,6-tetramethyl homologue (Tart) or Boc, which are onlycleaved under strongly acidic conditions. Preferably, Pbf, Pmc, Mtr,most preferably Pbf is used. Upon global deprotection of side chainsunder strongly acidic conditions, in usually aequeous medium,bystander-alkylation of deprotected tyrosine is not observed with Pmc,Mtr and Pbf. Serine and, Threonine typically may be protected by e.g.tert-butyl or trityl, most preferably tert-butyl. Other modes ofprotection are equally feasible, e.g. with benzyl, though less preferredsince eventually requiring removal under less desirable condition.Similar considerations apply to protection of Lysine; typically andpreferably, Lys is protected with Boc. Tryptophan must not necessarilybe protected during solid-phase synthesis, though protection withtypically Boc is evisaged. As regards side chain protection groups, theaforementioned is valid both for the natural L-amino acids as well asfor their D-homologues.

Therefore, in various embodiments, step (a) comprises the steps of:

-   -   (i) deprotecting a first amino acid linked to the solid support        by removing protective chemical groups from the first amino        acid;    -   (ii) activating chemical groups on a second amino acid to        prepare the second amino acid for coupling with the first amino        acid;    -   (iii) coupling the activated second amino acid to the        deprotected first amino acid to form a peptide from the first        and second amino acids; and    -   (iv) successively deprotecting, and coupling a plurality of        amino acids into the growing peptide chain until the ginsentide        or ginsentide-like peptide is synthesized.

The abundance of glycine and cysteine residues in the ginsentidesequence usually poses synthetic challenges in the chemical synthesisthereof, especially in the C-terminus. Attempts to synthesizeginsentides by stepwise synthesis using solid-phase methods have failedbecause the C-terminal fragments formed aggregates, preventing asuccessful synthesis. The method described herein addresses thesechallenges. In particular, a strategically selected glycine residue,i.e. the one at the position corresponding to position 24 of SEQ IDNO:1, is alkylated by a reversible protecting group to preventaggregation during the synthesis. Fmoc-(Dmb)Gly-OH is used for theaddition of the glycine residue at this position, while Fmoc-Gly-OH maybe used for the introduction of glycine residues at other positions.

Coupling reagents for Fmoc peptide synthesis are well-known in the art.Coupling reagents may be mixed anhydrides, (e.g. propane phosphonic acidanhydride) or other acylating agents such as activated esters or acidhalogenides (e.g. isobutyl-chloroformiate or ‘ICBF’), or they may becarbodiimides (e.g. 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide,diisopropyl-carbodiimide, dicylcohexyl-carbodiimide), activatedbenzotriazine-derivatives (e.g.3-(diethoxyphosphoryloxy)-1,2,3-benzotriazine-4(3H)-one or ‘DEPBT’) oruronium or phosphonium salt derivatives of benzotriazol. In view of bestyield, short reaction time and protection against racemization duringchain elongation, it is preferred that the coupling reagent is selectedfrom the group consisting of uronium salts and phosphonium salts ofbenzotriazol capable of activating a free carboxylic acid function alongwith that the reaction is carried out in the presence of a base.Suitable and likewise preferred examples of such uronium or phosphoniumcoupling salts are e.g. HBTU(O-1H-benzotriazole-1-yl)-N,N,N′,N′-tetramethyl uroniumhexafluorophosphate), BOP(benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate), PyBOP(Benzotriazole-1-yl-oxy-tripyrrolidinophosphonium hexafluorophosphate),PyAOP, HCTU(O-(1H-6-chloro-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate), TCTU (O-1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate), HATU(O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate), TATU(O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate), TOTU(O-[cyano(ethoxycarbonyl)methyleneamino]-N,N,N′,N′-tetramethyluroniumtetrafluoroborate), HAPyU(O-(benzotriazol-1-yl)oxpis-(pyrrolidino)-uronium hexafluorophosphate.

For coupling of the Fmoc amino acids to the peptide, the carboxyl groupis usually activated. This is important for speeding up the reaction.There are two main types of activating groups: carbodiimides andtriazolols. The use of these activating coupling additives isparticularly preferred when using the highly activating uronium orphosphonium salt coupling reagents. Most preferably the couplingadditive is a N-hydroxy-benzotriazol derivative (or1-hydroxy-benzotriazol derivative) or is an N-hydroxy-benzotriazinederivative. Suitable examples include N-hydroxy-succinimide,N-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HOOBt),1-hydroxy-7-azabenzotriazole (HOAt) and N-hydroxy-benzotriazole (HOBt).N-hydroxy-benzotriazine derivatives are particularly preferred, in amost preferred embodiment, the coupling reagent additive ishydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine. Most common carbodiimidesare dicyclohexylcarbodiimide (DCC) and diisopropylcarbodiimide (DIC).

Activation of the Fmoc amino acid is typically done in the presence of abase reagent. Preferably, the base reagent is a weak base whoseconjugated acid has a pKa value of from pKa 7.5 to 15, more preferablyof from pKa 7.5 to 10, and which base preferably is a tertiary,sterically hindered amine. Examples of such and further preferred areHunig-base (N,N-diisopropylethylamine; DIPEA), N,N′-dialkylaniline,2,4,6-trialkylpyridine, 2,6-trialkylpyridine or N-alkyl-morpholine withthe alkyl being straight or branched C₁-C₄ alkyl, more preferably it isN-methylmorpholine (NMM) or collidine (2,4,6-trimethylpyridine), mostpreferably it is collidine.

The amount of the various reactants in the coupling reaction can andwill vary greatly. Reagents are typically used in large excess tospeed-up the reaction and drive it to completion. Typically the amountof solid support to the amount of Fmoc-amino acid will be a molar ratioranging from about 1:1 to 1:10. In one embodiment, the amount of solidsupport to the amount of Fmoc-amino acid to the amount of activatingcompound is a molar ratio of about 1:4. The reaction conditions for thecoupling steps, such as reaction time, temperature, and pH may varywithout departing from the scope of the invention. The couplingtemperature is usually in the range of from 15 to 30° C., especiallywhere using phosphonium or uronium type coupling reagents. Typically, atemperature of about 20 to 25° C. is applied for coupling.

In various embodiments, the method comprises accelerating at least oneof the deprotecting, activating, and coupling steps by applyingmicrowave energy.

Microwave energy applied to the contents of the reaction vessel duringthe deprotecting, activating, coupling, and cleaving steps greatlydecreases the length of time necessary to complete these reactions. Themethod for applying microwave energy may be moderated by the microwavesource in such a way as to provide the fastest reaction time whileaccumulating the least amount of heat, thus more microwave energy may beapplied and heat-associated degradation of the reaction contents doesnot occur. This method may include, but not limited to, spiking themicrowave energy in large amounts for short lengths of time.

The method may include cooling the reaction vessel and thus itscontents, during and between applications of microwave energy up to andincluding the final cleaving step. The cooling mechanism of the methodoperates during amino acid extension cycles, the term “cycle” usedherein to refer to the deprotection, activation, and coupling necessaryto link one amino acid to another. The cooling system can also operateduring and between applications of microwave energy in a given cycle tokeep the bulk temperature of the reaction contents down. The coolingsystem can also operate when the complete peptide is cleaved from theresin. Alternatively, by controlling the power rather than strictlycontrolling the temperature, a desired control over the progress of areaction can also be provided.

It should be noted that the amino acid sequence of any one of SEQ IDNOs:1-64 as described above only defines the primary structure of theginsentide or ginsentide-like peptide, i.e. the sequence of amino acids,without including any information about the disulfide bridges (—S—S—)that may be existing in the peptide.

Disulfide bridges are produced by the oxidative folding of two differentthiol groups (—SH) present in the peptide. The peptide described hereincontains eight different thiol groups (i.e. eight cysteine residues eachcontaining a thiol group) and therefore may form zero, one, two, three,or four intramolecular disulfide bridges. The number of possibledisulfide bridge connectivity patterns (cysteine pairings) increasesrapidly with the number of bound cysteines.

To form native intracellular disulfide bonds, the synthesis processdescribed herein is followed by oxidative folding of the synthesizedpeptide. The oxidative folding of a peptide or a protein refers to theconcurrent formation of one or more native disulfide bonds of itsreduced form to an oxidized folded form with identical disulfideconnectivity with the native molecule. Disulfides in a peptide or andprotein stabilizes its structure and maintains its biologicalactivities. Many bioactive peptides are cysteine-rich, containing 2-5disulfide bonds. However, the oxidative folding or refolding ofsynthetic or recombinant peptides or proteins, particularly those thatare cysteine-rich, is invariably performed in an aqueous bufferedsolution and in the presence of a pair of reducing and oxidizingreagents to facilitate the correct disulfide bond formation bydisulfide-exchange reactions. However, oxidative folding in an aqueoussolution is a limiting step in their preparation because ofunpredictability of generating isomeric and dead-end products.Chemoselective disulfide approaches which selectively form eachdisulfide pair stepwise can achieve correct pairing disulfide bonds, butthe process is laborious and generally suffers from low yields.Approaches of oxidative folding for cysteine-rich peptides include airoxidation, the use of strong oxidizing agents or reduced/oxidizedglutathiones and invariably in aqueous media at a basic pH, which may bereferred to as aqueous oxidative folding. In this invention, anultra-fast oxidative folding of peptides in non-aqueous conditions andperformed in organic solvents (organic oxidative folding) has beenachieved. Compared to the aqueous oxidative folding, the organicoxidative folding is remarkably faster.

In various embodiments, organic oxidative folding comprises folding theginsentide or ginsentide-like peptide in an organic solvent (preferablyDMSO and/or isopropanol) comprising cysteamine and/or morpholine, e.g.in a folding buffer comprising 10% (v/v) DMSO, 84.36% (v/v) isopropanol,5% (v/v) morpholine and 0.64% (v/v) Cysteamine (10M).

In various embodiments, the organic oxidative folding is performed at 4°C.

It is believed that the method of organic oxidative folding describedherein enables the formation of native disulfide bonds of cysteine-richpeptides within minutes, e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, or 59 minutes, preferably within 5minutes, which is remarkably faster than conventional methods known inthe art such as aqueous oxidative folding.

In various embodiments, the method comprises purifying the foldedginsentide or ginsentide-like peptide or salt thereof, preferably byRP-HPLC.

Without wishing to be bound to any particular theory, it is believedthat the ginsentide or ginsentide-like peptide prepared by the methoddescribed herein have four native disulfide bonds as described inTable 1. The prepared ginsentide or ginsentide-like peptide is identicalto the natural peptide and is functionally indistinguishable therefrom.

It should be noted that the methods of solid-phase peptide synthesis andorganic oxidative folding described herein can also be used in thepreparation of other cysteine-rich peptides. Such uses are also withinthe scope of the present application.

In a second aspect, the invention relates to a method of recombinantlypreparing a ginsentide or ginsentide-like peptide or a pharmaceuticallyacceptable salt thereof, said ginsentide or ginsentide-like peptidehaving (i) the amino acid sequence set forth in any one of SEQ IDNO:1-64; or (ii) the amino acid sequence sharing at least 65%,preferably at least 75%, even more preferably at least 85%, mostpreferably at least 95% sequence identity with the peptide of (i) overits entire sequence, wherein the method comprises the steps of:

-   -   (a) providing a host cell comprising a polynucleotide, wherein        the polynucleotide encodes a polypeptide comprising        Maltose-binding protein having the amino acid sequence set forth        in SEQ ID NO:65, a enterokinase cleavage sequence having the        amino acid sequence set forth in SEQ ID NO:66, and the        ginsentide or ginsentide-like peptide or salt thereof;    -   (b) culturing the host cell in a growth medium under conditions        allowing production of the polypeptide, and recovering the        polypeptide from the medium; and    -   (c) generating the ginsentide or ginsentide-like peptide or salt        thereof by cleaving the polypeptide using enterokinase.

The polypeptide may be recombinantly expressed in a host cell or acell-free system. The polypeptide may be produced using any known andwell-established expression system and recombinant cell culturingtechnology. The polypeptide may also be produced in a host cell in atransgenic organism such as a goat or a plant.

The term “host cell” for the purposes of the present invention refers toany cell that is commonly used for expression, i.e. transcription andtranslation of the polynucleotides for the production of thepolypeptides of interest. In particular, the term “host cell” relates toprokaryotes, lower eukaryotes, plants, insect cells or mammalian cellculture systems. Host cells include, without limitation, bacterial,microbial, plant or animal cells, e.g. Escherichia coli, Bacillussubtilis; Saccharomyces cerevisiae, Pichia pastoris, or CHO cells.

For recombinant production of the polypeptide typically a polynucleotideencoding the polypeptide is isolated and inserted into a replicablevector such as a plasmid for further cloning (amplification) orexpression. In various embodiments a polynucleotide encoding apolypeptide according to the invention is included in a vector such as aplasmid. The term “vector” as used herein refers to a polynucleotidecapable of transporting another nucleic acid to which it has beenlinked. In one embodiment, the vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. In another embodiment, the vector is a viral vector, whereinadditional DNA segments may be ligated into the viral genome. Thevectors disclosed herein can be capable of autonomous replication in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication and episomal mammalian vectors) or canbe can be integrated into the genome of a host cell upon introductioninto the host cell, and thereby are replicated along with the hostgenome (e.g., non-episomal mammalian vectors).

When using recombinant techniques, the polypeptide can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the polypeptide is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, areremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 1992; 10: 163-167 describe a procedure for isolatingpolypeptides which are secreted to the periplasmic space of E coli. Thepolypeptide can either be directly obtained in a soluble and foldedstate or recovered in form of inclusion bodies, followed by renaturationin vitro. A further option is the use of specific host strains having anoxidizing intracellular milieu, which may thus allow the formation ofdisulfide bonds in the cytosol (Venturi M, Seifert C, Hunte C. J MolBiol 2002; 315, 1-8).

In various embodiments, the polypeptide has the amino acid sequence setforth in any one of SEQ ID NOs: 67-130.

TABLE 3 Amino acid sequences of SEQ ID NOs: 65-80 Name SEQ ID NOSequence Maltose-binding 65 MKIKTGARILALSALTTMMFSASALAKIEEGKLVIW protein INGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEK FPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTVVDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTVVEEIPALDKELKAKGKSALMFNLQEPYFTVVPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFVVYAVRTAVINAASGRQTVDEALKDAQT NSSSNNNNNNNNNNLGenterokinase cleavage 66 DDDDK sequence Maltose-binding 67MKIKTGARILALSALTTMMFSASALAKIEEGKLVIW  protein-enterokinaseINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEK  cleavage sequence-FPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPD  TP1KAFQDKLYPFTVVDAVRYNGKLIAYPIAVEALSLIY NKDLLPNPPKTVVEEIPALDKELKAKGKSALMFNLQEPYFTVVPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFVVYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGDDDDKCKSGGAWCGFDPHGC CGNCGCLVGFCYGTGC Maltose-binding 68MKIKTGARILALSALTTMMFSASALAKIEEGKLVIW  protein-enterokinaseINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEK cleavage sequence-FPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPD TP2KAFQDKLYPFTVVDAVRYNGKLIAYPIAVEALSLIY NKDLLPNPPKTVVEEIPALDKELKAKGKSALMFNLQEPYFTVVPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFVVYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGDDDDKCKSSGAWCGFDPHGC CGNCGCLVGFCYGTGC Maltose-binding 69MKIKTGARILALSALTTMMFSASALAKIEEGKLVIW  protein-enterokinaseINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEK  cleavage sequence-FPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPD  TP3KAFQDKLYPFTVVDAVRYNGKLIAYPIAVEALSLIY NKDLLPNPPKTVVEEIPALDKELKAKGKSALMFNLQEPYFTVVPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFVVYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGDDDDKCKSAGTWCGFDPHGC CGSCGCLVGFCYGVSC Maltose-binding 70MKIKTGARILALSALTTMMFSASALAKIEEGKLVIW protein-enterokinaseINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEK cleavage sequence-FPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPD TP4KAFQDKLYPFTVVDAVRYNGKLIAYPIAVEALSLIY NKDLLPNPPKTVVEEIPALDKELKAKGKSALMFNLQEPYFTVVPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFVVYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGDDDDKCLKNGEFCWGDPSGC CGNCGCLIIPGVCYGTGC Maltose-binding71 MKIKTGARILALSALTTMMFSASALAKIEEGKLVIW  protein-enterokinaseINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEK  cleavage sequence-FPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPD  TP5KAFQDKLYPFTVVDAVRYNGKLIAYPIAVEALSLIY NKDLLPNPPKTVVEEIPALDKELKAKGKSALMFNLQEPYFTVVPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFVVYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGDDDDKCKSSGAWCGFDPHGC CGNCGCLVGFCYGTDC Maltose-binding 72MKIKTGARILALSALTTMMFSASALAKIEEGKLVIW protein-enterokinaseINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEK cleavage sequence-FPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPD TP6KAFQDKLYPFTVVDAVRYNGKLIAYPIAVEALSLIY NKDLLPNPPKTVVEEIPALDKELKAKGKSALMFNLQEPYFTVVPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFVVYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGDDDDKCIPGGGFCMFEPLSC CVNCGCILVPGVCYCG Maltose-binding 73MKIKTGARILALSALTTMMFSASALAKIEEGKLVIW protein-enterokinaseINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEK cleavage sequence-FPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPD TP7KAFQDKLYPFTVVDAVRYNGKLIAYPIAVEALSLIY NKDLLPNPPKTVVEEIPALDKELKAKGKSALMFNLQEPYFTVVPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFVVYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGDDDDKCKSGGTWCGFDPHGC CGNCGCLVGFCYGTGC Maltose-binding 74MKIKTGARILALSALTTMMFSASALAKIEEGKLVIW protein-enterokinaseINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEK cleavage sequence-FPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPD TP8KAFQDKLYPFTVVDAVRYNGKLIAYPIAVEALSLIY NKDLLPNPPKTVVEEIPALDKELKAKGKSALMFNLQEPYFTVVPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFVVYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGDDDDKCISSGGWCGFDLHGC CGNCGCLVGFCYGTGC Maltose-binding 75MKIKTGARILALSALTTMMFSASALAKIEEGKLVIW  protein-enterokinaseINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEK  cleavage sequence-FPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPD  TP9KAFQDKLYPFTVVDAVRYNGKLIAYPIAVEALSLIY NKDLLPNPPKTVVEEIPALDKELKAKGKSALMFNLQEPYFTVVPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFVVYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGDDDDKCKSGGSWCGFDPHGC CGNCGCLVGFCYGTGC Maltose-binding 76MKIKTGARILALSALTTMMFSASALAKIEEGKLVIW  protein-enterokinaseINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEK  cleavage sequence-FPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPD  TP10KAFQDKLYPFTVVDAVRYNGKLIAYPIAVEALSLIY NKDLLPNPPKTVVEEIPALDKELKAKGKSALMFNLQEPYFTVVPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFVVYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGDDDDKCIFSGGWCGFDLHGC CGNCGCLVGFCYGTGC Maltose-binding 77MKIKTGARILALSALTTMMFSASALAKIEEGKLVIW  protein-enterokinaseINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEK  cleavage sequence-FPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPD  TP11KAFQDKLYPFTVVDAVRYNGKLIAYPIAVEALSLIY NKDLLPNPPKTVVEEIPALDKELKAKGKSALMFNLQEPYFTVVPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFVVYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGDDDDKCLKNGQFCWGNPSGC CGNCGCLIIPGVCYGTGC Maltose-binding78 MKIKTGARILALSALTTMMFSASALAKIEEGKLVIW  protein-enterokinaseINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEK  cleavage sequence-FPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPD  TP12KAFQDKLYPFTVVDAVRYNGKLIAYPIAVEALSLIY NKDLLPNPPKTVVEEIPALDKELKAKGKSALMFNLQEPYFTVVPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFVVYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGDDDDKCIPGGGFCMFEPLSC CHNCGCLLVPGVCYCG Maltose-binding 79MKIKTGARILALSALTTMMFSASALAKIEEGKLVIW  protein-enterokinaseINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEK  cleavage sequence-FPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPD  TP13KAFQDKLYPFTVVDAVRYNGKLIAYPIAVEALSLIY NKDLLPNPPKTVVEEIPALDKELKAKGKSALMFNLQEPYFTVVPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFVVYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGDDDDKCIPNGGFCMFEPLSC CVNCGCILVPGVCYCG Maltose-binding 80MKIKTGARILALSALTTMMFSASALAKIEEGKLVIW  protein-enterokinaseINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEK  cleavage sequence-FPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPD  TP14KAFQDKLYPFTVVDAVRYNGKLIAYPIAVEALSLIY NKDLLPNPPKTVVEEIPALDKELKAKGKSALMFNLQEPYFTVVPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFVVYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGDDDDKCLKVGKICLGRGLKE CCPSATCGCLLGFCIKC

The recombinantly produced polypeptide is further cleaved byenterokinase immediately after the C-terminal ends of the enterokinasecleavage site (Asp-Asp-Asp-Asp-Lys, SEQ ID NO:66) such that theginsentide or ginsentide-like peptide is released for furtherpurification.

In various embodiments, the method further comprises purification of therecombinantly produced polypeptide prior to the enterokinase cleavage,preferably by amylose resin purification.

The polypeptide produced by the cells can be purified using anyconventional purification technology, for example, by gelelectrophoresis, dialysis, affinity chromatography, or preferably byanion-exchange chromatography and/or reverse-phase chromatography. Thechoice of the purification method that is used for a particularpolypeptide of the invention is within the knowledge of the person ofaverage skill in the art.

Without wishing to be bound to any particular theory, ginsentide orginsentide-like peptides recombinantly produced using the methoddescribed herein have native disulfide bonds making organic oxidativefolding of the ginsentide or ginsentide-like peptides unnecessary.

Due to their high sequence similarity, it is believed that theginsentides or ginsentide-like peptides described herein have similarbiological functions.

In addition, it should be understood that a polyptide comprising,consisting essentially of, or consisting of a ginsentide orginsentide-like peptide of the invention may also be prepared using themethods detailed above. It may also be used in the applicationsdescribed below. Such methods of preparation and uses of thesepolypeptides are also within the scope of the present application. Theginsentide or ginsentide-like peptides and the polypeptides describedherein are preferably substantially free of impurities, but may stillcontain extraneous compounds or impurities that can come from manysources, such as unreacted starting materials, by-products, degradationproducts, and residual components of host cells.

It should also be understood that enriched fractions obtained bychromatographic, size-exclusion, or methods related to purification toenrich peptides with MW between 2-5 kDa from ginseng plants or plantscontaining ginsentide-related compounds from natural sources, as well astheir use in the nutraceutical, pharmaceutical or cosmetic applicationsdescribed below are also within the scope of the present application.

In a third aspect, the invention relates to use of a ginsentide orginsentide-like peptide or a pharmaceutically acceptable salt thereof orenriched fractions by chromatographic, size-exclusion, or methodsrelated to purification from ginseng plants or plants containingginsentides or ginsentide-like peptides thereof, said ginsentide orginsentide-like peptide having (i) the amino acid sequence set forth inany one of SEQ ID NO:1-64; or (ii) the amino acid sequence sharing atleast 65%, preferably at least 75%, even more preferably at least 85%,most preferably at least 95% sequence identity with the peptide of (i)over its entire sequence, as an al-adrenergic receptor antagonist andvasorelaxant.

In a fourth aspect, the invention relates to use of a ginsentide orginsentide-like peptide or a pharmaceutically acceptable salt thereof orenriched fractions by chromatographic, size-exclusion, or methodsrelated to purification from ginseng plants or plants containingginsentides or ginsentide-like peptides thereof, said ginsentide orginsentide-like peptide having (i) the amino acid sequence set forth inany one of SEQ ID NO:1-64; or (ii) the amino acid sequence sharing atleast 65%, preferably at least 75%, even more preferably at least 85%,most preferably at least 95% sequence identity with the peptide of (i)over its entire sequence, as a nitric oxide-boosting agent.

In a fifth aspect, the invention relates to use of a ginsentide orginsentide-like peptide or a pharmaceutically acceptable salt thereof orenriched fractions by chromatographic, size-exclusion, or methodsrelated to purification from ginseng plants or plants containingginsentides or ginsentide-like peptides thereof, said ginsentide orginsentide-like peptide having (i) the amino acid sequence set forth inany one of SEQ ID NO:1-64; or (ii) the amino acid sequence sharing atleast 65%, preferably at least 75%, even more preferably at least 85%,most preferably at least 95% sequence identity with the peptide of (i)over its entire sequence, as an anti-thrombotic agent.

In a sixth aspect, the invention relates to use of a ginsentide orginsentide-like peptide or a pharmaceutically acceptable salt thereof orenriched fractions by chromatographic, size-exclusion, or methodsrelated to purification from ginseng plants or plants containingginsentides or ginsentide-like peptides thereof, said ginsentide orginsentide-like peptide having (i) the amino acid sequence set forth inany one of SEQ ID NO:1-64; or (ii) the amino acid sequence sharing atleast 65%, preferably at least 75%, even more preferably at least 85%,most preferably at least 95% sequence identity with the peptide of (i)over its entire sequence, as an anti-atherosclerotic agent.

In a seventh aspect, the invention relates to use of a ginsentide orginsentide-like peptide or a pharmaceutically acceptable salt thereof orenriched fractions by chromatographic, size-exclusion, or methodsrelated to purification from ginseng plants or plants containingginsentides or ginsentide-like peptides thereof, said ginsentide orginsentide-like peptide having (i) the amino acid sequence set forth inany one of SEQ ID NO:1-64; or (ii) the amino acid sequence sharing atleast 65%, preferably at least 75%, even more preferably at least 85%,most preferably at least 95% sequence identity with the peptide of (i)over its entire sequence, as a protective agent againstdoxorubicin-induced cardiotoxicity.

In an eighth aspect, the invention relates to use of a ginsentide orginsentide-like peptide or a pharmaceutically acceptable salt thereof orenriched fractions by chromatographic, size-exclusion, or methodsrelated to purification from ginseng plants or plants containingginsentides or ginsentide-like peptides thereof, said ginsentide orginsentide-like peptide having (i) the amino acid sequence set forth inany one of SEQ ID NO:1-64; or (ii) the amino acid sequence sharing atleast 65%, preferably at least 75%, even more preferably at least 85%,most preferably at least 95% sequence identity with the peptide of (i)over its entire sequence, as an anti-ageing and adaptogenic agent.

In a ninth aspect, the invention relates to use of a ginsentide orginsentide-like peptide or a pharmaceutically acceptable salt thereof orenriched fractions by chromatographic, size-exclusion, or methodsrelated to purification from ginseng plants or plants containingginsentides or ginsentide-like peptides thereof, said ginsentide orginsentide-like peptide having (i) the amino acid sequence set forth inany one of SEQ ID NO:1-64; or (ii) the amino acid sequence sharing atleast 65%, preferably at least 75%, even more preferably at least 85%,most preferably at least 95% sequence identity with the peptide of (i)over its entire sequence, as a nutraceutical, a health supplement, or acosmetic ingredient.

The term “nutraceutical” as used herein refers to a substance intendedto supplement a diet and provide nutrients, such as, for example,vitamins, minerals, fiber, fatty acids, or amino acids, that may bemissing or may not be consumed in sufficient quantity in the diet. Theterm “health supplement” as used herein includes food and foodsupplement to animals and/or humans, fortification of food, dietarysupplement, functional (and medical) food and nutrient supplement. Theterm “cosmetic ingredient” as used herein refers to any agent that maybe comprised in a cosmetic formulation for cosmetic use, such as skinconditioning agents and anti-aging agents.

In various embodiments of the afore-described third to ninth aspects ofthe invention, the ginsentide or ginsentide-like peptide or saltthereof, regardless of whether it is commercially obtained, isolatedfrom natural sources, or preferably synthetically or recombinantlyprepared by a method described herein, can be used in vitro as abiologically active agent. In various embodiments, it can beadministered to a subject (e.g. a human or an animal) in need thereof inan effective amount for the treatment or prevention of a condition,disease or disorder considered suitable, alone or in combination(simultaneously, sequentially or separately) with one or more otheragents. Such conditions, diseases or disorders include, withoutlimitation, hypertension, ischaemic heart disease, thrombosis,atherosclerosis, erectile dysfunction, stroke, myocardial infarction,heart failure, cancer, aging and aging-related diseases.

Accordingly, the ginsentide or ginsentide-like peptide or salt thereofcan be formulated into compositions further comprising apharmaceutically acceptable carrier. The term “pharmaceuticallyacceptable carrier” as used herein means a pharmaceutically acceptablematerial, composition or vehicle, such as a liquid or solid filler,diluent, excipient, or solvent encapsulating material, involved incarrying or transporting the peptide from one organ, or portion of thebody, to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not injurious to the patient. Some examples ofmaterials which can serve as pharmaceutically acceptable carriersinclude: sugars, such as lactose, glucose and sucrose; starches, such ascorn starch and potato starch; cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients, such as cocoabutter and suppository waxes; oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols,such as propylene glycol; polyols, such as glycerin, sorbitol, mannitoland polyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents, such as magnesium hydroxide and aluminumhydroxide; alginic acid; sterile distilled water; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol; pH bufferedsolutions; polyesters, polycarbonates or polyanhydrides; and othernon-toxic compatible substances employed in pharmaceutical formulations.See Remington: The Science and Practice of Pharmacy, 19th Ed. (Easton,Pa.: Mack Publishing Co., 1995), which discloses typical carriers andconventional methods of preparing pharmaceutical formulations.

The skilled artisan would also realize that proper formulation isdependent upon the route of administration selected for the specificapplication, and the proper route and mode of administering the peptideof the invention to a subject should be determined on a case-by-casebasis.

The composition of the invention can be administered via any parenteralor non-parenteral (enteral) route that is therapeutically effective forproteinaceous or nucleic acid-based drugs. Parenteral applicationmethods include, for example, intracutaneous, subcutaneous,intramuscular, intratracheal, intranasal, intravitreal or intravenousinjection and infusion techniques, e.g. in the form of injectionsolutions, infusion solutions or tinctures, as well as aerosolinstallation and inhalation, e.g. in the form of aerosol mixtures,sprays or powders. An overview about pulmonary drug delivery, i.e.either via inhalation of aerosols (which can also be used in intranasaladministration) or intracheal instillation is given by Patton et al.Proc Amer Thoracic Soc 2004; Vol. 1 pages 338-344, for example).Non-parenteral delivery modes are, for instance, orally, e.g. in theform of pills, tablets, capsules, solutions or suspensions, or rectally,e.g. in the form of suppositories. Peptides of the invention can beadministered systemically or topically in formulations containingconventional non-toxic pharmaceutically acceptable excipients orcarriers, additives and vehicles as desired.

In one embodiment of the present invention the pharmaceutical isadministered parenterally to a mammal, and in particular to humans.Corresponding administration methods include, but are not limited to,for example, intracutaneous, subcutaneous, intramuscular, intratrachealor intravenous injection and infusion techniques, e.g. in the form ofinjection solutions, infusion solutions or tinctures as well as aerosolinstallation and inhalation, e.g. in the form of aerosol mixtures,sprays or powders.

The dosage of the peptide of the invention applied may vary within widelimits to achieve the desired preventive effect or therapeutic response.It will, for instance, depend on the half-life of the peptide in vivo.Further, the optimal dosage will depend on the biodistribution of thepeptide, the mode of administration, the severity of thedisease/disorder being treated as well as the medical condition of thepatient. If wanted, the peptide may also be given in a sustained releaseformulation, for example liposomal dispersions or hydrogel-based polymermicrospheres, like PolyActive™ or OctoDEX™ (cf. Bos et al., BusinessBriefing: Pharmatech 2003: 1-6). Other sustained release formulationsavailable are for example PLGA based polymers (PR pharmaceuticals),PLA-PEG based hydrogels (Medincell) and PEA based polymers (Medivas).

Accordingly, the peptide of the present invention can be formulated intocompositions using pharmaceutically acceptable ingredients as well asestablished methods of preparation (Gennaro, A. L. and Gennaro, A. R.(2000) Remington: The Science and Practice of Pharmacy, 20th Ed.,Lippincott Williams & Wilkins, Philadelphia, Pa.). To prepare thepharmaceutical compositions, pharmaceutically inert inorganic or organicexcipients can be used. To prepare e.g. pills, powders, gelatinecapsules or suppositories, for example, lactose, talc, stearic acid andits salts, fats, waxes, solid or liquid polyols, natural and hardenedoils can be used. Suitable excipients for the production of solutions,suspensions, emulsions, aerosol mixtures or powders for reconstitutioninto solutions or aerosol mixtures prior to use include water, alcohols,glycerol, polyols, and suitable mixtures thereof as well as vegetableoils.

In some embodiments, the ginsentide or ginsentide-like peptide or saltthereof can be comprised in any cosmetic compositions known in the artto provide for topical skin rejuvenation and/or anti-aging compositions.

The formulations can be sterilized by numerous means, includingfiltration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile medium justprior to use.

The compositions and the methods of therapeutic or commetic applicationsof the ginsentide or ginsentide-like peptides and compositions describedabove are also within the scope of the present application.

The present invention is further illustrated by the following examples.However, it should be understood, that the invention is not limited tothe exemplified embodiments.

EXAMPLES Materials and Methods General Procedures for Peptide SolidPhase Synthesis by Fmoc-Chemistry

For peptide-hydrazides, firstly, the 2-chlorotritylchloride resin wasswelled in dry DCM for a while, 5% hydrazine in dry DMF was directlyadded. After 0.5 hr, the resin was washed with DCM (3×), DMF (3×), andDCM (3×). Peptides were then assembled on the hydrazine-trityl resinusing typical Fmoc SPPS protocols. All amino acids were used in 4 eq ofthe resin, and preactivated by 4 eq of PyBop and 8 eq of DIEA. After 1to 2 hr coupling of amino acid, the resin was washed with DCM (3×), DMF(3×), and DCM (3×). The successive a-amino group deprotection wasperformed in 20% piperidine in DMF (5 min, 10 min) between each aminoaicd coupling step. Finally, a peptide-hydrazide was released from resinby using the cleavage reagent: a mixture of 90% TFA, 5% EDT, 2.5% TISand 2.5% water. After precipitated from ether, the crudepeptide-hydrazide was purified by prep-HPLC, the molecular weight wasdetermined by ESI-MS.

For cysteinyl peptides, firstly, 2 eq of Fmoc-Cys(Trt)-OH was dissolvedin dry DCM (with a small amount of dry DMF for solubility), and addedinto the swelled 2-chlorotritylchloride resin with 5 eq (respective tothe amino acid) of DIEA. After 1 hr reaction, the resin was washed withDCM (3×), DMF (3×), and DCM (3×). For capping any remaining reactivetrityl groups, the resin was reacted with methanol for 15 min. Once thecysteine residue was attached on the resin, amino acid coupling, α-aminogroup deprotection and final peptide cleavage were performed accordingto the same procedure described in the above.

Microwave-Assisted Automated Peptide Synthesis

Microwave-assisted peptide synthesis was performed in 0.1 mmol and 0.25mmol on CEM microwave peptide synthesizer, liberty 1 according to themanual instruction. Fmoc-(Dmb) Gly-OH was used in peptide C-terminalpart instead of Fmoc-Gly-OH. Fmoc deprotection was done in 20%piperidine/DMF containing 0.1 M HOBt. Coupling of Fmoc/tBu-protectedamino acids were achieved by 5 eq amino acids activated by PyBop in DMF.The parameters used in the microwave synthesis was shown in the belowtable.

TABLE 6 MW-SPPS parameters used in the synthesis (CEM microwave peptidesynthesizer, liberty 1) Number of Amino acid couplings Power (W) Temp (°C.) Time (s) Coupling Asn 2 20 50 600 Asp 2 20 50 600 Cys 2 20 50 600Pro 2 20 50 600 AA followed by 1 20 50 600 Pro Other AAs DeprotectionInitial depro 1 20 55 30 depro 1 20 55 180

After the completion of peptide synthesis, the peptide was cleaved in amixture of 90% TFA, 5% EDT, 2.5% TIS and 2.5% water. And then, thepeptide precipitated by ether was directly used in the folding reaction.

Ligation of Peptide-Hydrazide with Cysteinyl Peptide

Briefly, a Fmoc-peptide hydrazide was first oxidized to form peptideazide by sodium nitrite (5 eq of peptide) at pH 3.0 in 0.2 M phosphatebuffer, pH 3.0, containing 6 M Gdn-HCl, at −20 degree for 20 min. Afteraddition of sodium 2-mercaptoethanesulfonate (MESNa) (final conc. 1%),and adjustment of pH to 7.0, the peptide thioester was then formed.Finally, this peptide thioester reacted chemoselectively withCysteinyl-peptide to produce the full-length peptide by generating anative amide bond at the ligation site. The removal of Fmoc from theligated full-length peptide was done in 20% piperidine with TCEP for 20min.

Folding of Reduced Peptides to Native Form

For most ginsentides and cysteine-rich peptides, the folding wasperformed in 20% DMSO in 0.1 M (NH)₄HCO₃ containingcysteamine/cystamine. The ratio of peptide: cysteamine:cystamine wasaround 1:200:10. 10% TFE, 5% or 10% IPA may be included depending onpeptides' solubility. Peptides were dissolved in very small amount ofDMSO, and then, added into the folding buffer, folded at 4 degree,overnight. After folding, the reaction was buffer-exchanged andconcentrated by using Sek-Pak C18 cartridge. The concentrated foldingmixture was purified by using HPLC to get pure folded products.

For organic oxidative folding, peptides were dissolved in very smallamount of DMSO, added into the folding buffer comprising 10% (v/v) DMSO,84.36% (v/v) isopropanol, 5% (v/v) morpholine and 0.64% (v/v) Cysteamine(10M), and was folded at 4° C. for 5 min. After folding, the reactionwas buffer-exchanged and concentrated by using Sek-Pak C18 cartridge.The concentrated folding mixture was purified by using HPLC to get purefolded products.

Recombinant Expression of Ginsentides

Overall Strategies for the Recombinant Expression of Ginsentides

1. Targeted sequence was cloned into a vector, where the ginsentide wasfused to the C-terminal of periplasmic signal sequence-maltose bindingprotein (MBP) with a DDDK cleavable sequence in between (enterokinasecan cleave at the C-terminal of DDDK);

2. Plasmid was transformed into E. coli and selected usingampicillin-containing agar plates;

3. A single colony was picked and cultured in liters ofampicillin-containing LB broth at 37° C., shaking at 200 rpm untiloptical density reaches around 0.6;

4. Induction was performed using 0.1 mM IPTG at 16° C. overnight;

5. The cell lysate was loaded to an amylose affinity column and elutedusing maltose; and

6. Enterokinase was used for cleavage of maltose binding protein (MBP).

7. Anion exchange and reversed phase-HPLC was used to purify therecombinant expressed ginsentides.

Isolation and Purification of Ginsentides from Panax ginseng, P.quinquefolius, or P. notoginseng

About 1 kg of dried roots, leaves, seeds or flowers of Panax ginseng, P.quinquefolius, or P. notoginseng were extracted with 10 litres of water.After filtration, the filtrate was then loaded onto a C18 flash column(Grace Davison, US) and eluted with 70% ethanol. The eluted fractionswere then loaded onto an SP Sepharose resin column (GE Healthcare, UK),eluted with 1 M NaCl (pH 3.0), and followed by ultrafiltration (ViVaflow200, 2000 MWCO hydrostat). Further purification was performed byreversed-phase high performance liquid chromatography (RP-HPLC)(Shimadzu, Japan). A linear gradient of mobile phase A (0.05% TFA/H₂O)and mobile phase B (0.05% TFA/ACN) was used on the C18 column (250×22mm, 5 μm, 300 Å) (Grace Davison, US).

This invention relates to the synthesis and use of ginsentides as apeptidogenic adaptogen to prolong our health span. The role of peptideadaptogenics is to extend the chronological clock of cellular andorganismal ageing. In turn, they intervene age-related diseases andpromote active and healthy ageing. Ginsentides in particular, are ableto address cardiovascular diseases and frailty, which are the underlyingcause of numerous chronic and old-age-related diseases such as cancer,cardiovascular diseases, inflammatory conditions, stress, and othermetabolic conditions. FIG. 1 is a schematic diagram depicting theadaptogenic effects of ginsentide. The synthesis and uses of ginsentideswill be described in the following sections:

-   -   1. Ginsentides production    -   2. Ginsentides as α1-adrenergic receptor antagonists and        vasorelaxants of rat aorta ring    -   3. Ginsentides as nitric oxide-boosting agents    -   4. Ginsentides as anti-thrombotic agents    -   5. Ginsentides as anti-atherosclerotic agents    -   6. Ginsentides as protective agents against doxorubicin-induced        cardiotoxicity    -   7. Ginsentides as anti-ageing and adaptogenic agent

Example 1: Ginsentides Production

The abundance of glycine and cysteine in the ginsentide sequence posedsynthetic challenges in their synthesis of ginsentides, especially inthe C-terminus. Attempts to synthesize ginsentides by a stepwisesynthesis using solid-phase methods failed because the C-terminalfragments formed aggregates, preventing a successful synthesis.Therefore, the present invention described a stepwise solid-phasesynthesis strategies of the chemical synthesis of ginsentides toovercome these synthetic challenges. In particular, a strategicallyselected Gly residue was alkylated by a reversible protecting group toprevent aggregation to permit a successful synthesis of ginsentide TP1as a representative example.

An organic oxidative folding system for disulfide bond formation hasbeen invented. Using this invention, the oxidative folding isaccelerated a thousand fold in forming native disulfide bonds ofcysteine-rich peptides within minutes (e.g. within 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 59 minutes) instead ofhours. The invention also improves reaction yield and can be performedin high peptide concentrations without observable peptideprecipitations. In addition, all reagents used in this method areinexpensive, which makes the synthesis economically favorable. Thus, itcan be applied to large scale production of cysteine-rich peptides.

Recombinant expression of ginsentides is advantageous as the use ofchemicals for the production of ginsentides will be eliminated.Moreover, the expressed ginsentides produced using the recombinantexpression platform will be in its folded state, making the second stepof oxidative folding unnecessary.

Technical Description of the Invention

-   -   i. Table 1 illustrates the amino acid sequence of ginsentides.    -   ii. FIG. 2 shows the schematic flow for the chemical synthesis        of ginsentides    -   iii. FIG. 3 shows the oxidative folding strategies for synthetic        ginsentide TP1    -   iv. FIG. 4, 5, 6 shows that the synthetic ginsentide is        identical to natural ginsentides, and is functionally        indistinguishable from natural ginsentide obtained from ginseng.    -   v. FIG. 7-11 shows the recombinant expression scheme for        ginsentide.

Oxidative folding relates to an approach for forming disulfide bonds inpeptides and proteins. In this method, reduced or partially oxidizedpeptides are fully oxidized in a nonaqueous system using organicsolvents and compatible organic reducing and oxidizing (redox) reagents.

The rationale of our invention is based on the observation thatoxidative folding is mediated by thiol-disulfide exchange reactionswhich are SN2 reaction. Such reactions are strongly favored in organicsolvents than in aqueous conditions in which the attacking thiolnucleophiles are being solvated by water. Eliminating water in theoxidative folding condition should accelerate the thiol-disulfideexchange reactions, and in turn, the oxidative process.

In this invention, four major components are introduced: an organicbase, organic solvent, structure-enhancing co-solvent and redoxreagents. An organic base, such as pyridine, imidazole or morpholine, isused to provide a suitable basicity for the disulfide formation whileorganic solvent provides an environment in which the thiol-disulfideexchange reaction is one thousand times faster than in aqueousconditions, which are the prevailing practice in literature.

In addition, structure-enhancing co-solvents and redox reagents ensurethe correct disulfide paring of folding products. Usingstructure-enhancing solvents such as trifluoroethanol (TFE), the nativesecondary structure is formed preferentially, thus bringing the nativepairing of cysteines in proximity to form the correct disulfideconnections. Using organic-solvent-compatible reductant such ascysteamine and oxidant dimethyl sulfoxide (DMSO), the incorrectdisulfide connection is able to be shuffled to the correct disulfideconnection by thiol-disulfide exchange reactions.

The reduced peptides can be synthesized by either solid/solution phasesynthesis or recombinant expression. Then it is purified andlyophilized. Reduced peptides are dissolved in pure DMSO, which alsoacts as a mild oxidant, and diluted with organic solvents to a finalconcentration of 0.1 mM to 1 mM. Subsequently, an organic base andreductants are added. The reductant can be dissolved in a small amountof water if it is not soluble in organic solvents. It is preferred thatthe final volume of water should not exceed 20% of the whole mixture.

The aforesaid procedure is employed to fold a cyclic peptide kalata B1(kB1) as an example. Dissolve fully reduced kB1 in DMSO as a stock. Add2-propanol as the organic solvent to dilute the peptide stock. Then addorganic bases including pyridine, imidazole or morpholine and thereductant cysteamine. Each condition contains 100 μM pf reduced kB1, 10%(v/v) DMSO and 100 mM cysteamine with various concentrations of solventand base.

-   -   FIG. 12 shows the RP-HPLC and MALDI-TOF MS profiles of native        and reduced kB1.    -   Table 4 shows the oxidative folding conditions of kB1 with        varying solvent combinations.    -   FIG. 13 shows the RP-UPLC profiles of oxidative folding of kB1        showing the effect of different bases.    -   FIG. 14 shows the RP-UPLC profiles of oxidative folding of kB1        and the effect of cysteamine and DMSO as redox agents.    -   FIG. 15 shows the RP-HPLC profiles of oxidative folding of kB1        with different thiols as reducing reagents.    -   FIG. 16 compares the 1H NMR spectra comparison of the natural        and synthetic kB1 prepared by organic oxidative folding.

TABLE 4 Oxidative folding conditions of kB1 with varying solventconcentrations Cysteamine Peptide Base (v/v %) 2-propanol DMSO Conc.Conc. Yield* Run Pyridine Morpholine Imidazole (%) (%) (mM) (μM) (%)  1 5 85 10 100 100 27  2 30 60 10 100 100 72  3 50 40 10 100 100 82  4 90 0 10 100 100  8  5  5 85 10 100 100 80  6 10 80 10 100 100 83  7 15 7510 100 100 84  8  5 85 10 100 100 88  9 10 80 10 100 100 89 10 20 70 10100 100 91 11 30 60 10 100 100 90 *Yields are calculated accordinglybased on RP-HPLC profiles of reactions at 1 h.

Example 2. Ginsentides as α1 Adrenergic Receptor Antagonists andVasorelaxants of Rat Aorta Ring

The present invention shows that ginsentide TP1 has vasorelaxationproperties.

Plant peptides and proteins are often underexplored as bioactiveconstituents in medicinal plants. Unlike other peptides and proteins,multiple intramolecular disulfide bonds stabilize cysteine-rich peptidesmaking them extremely stable against heat, acid and enzymaticdegradation. The present invention shows that ginsentide TP1, acysteine-rich peptide, has eight cysteine residues (four stabledisulfide bonds) and a unique pseudo-cyclic structure, making it stableagainst heat, acid, serum and proteolytic degradation.

Cysteine-rich peptides are highly cross-linked by intramoleculardisulfide bridges. This forces the bulky hydrophobic side chains toexpose outward, making the surface of the molecule more lipophilic.Also, the slow exchange of D₂O indicates that the hydrogen bonding ofginsentides are intramolecularly connected instead of exposing outwards.These characteristics desolvate ginsentides from water. Therefore, theunique features of ginsentides could improve the intrinsic cellpermeability and intestinal absorption, making it orally active.

Technical Description

FIG. 17 shows a schematic purification of ginsentides from ginsengplants. All ginsentides including TP1 (herein refer as naturalginsentide TP1) were purified to high homogeneity throughsolvent-solvent partition, ion-exchange, size exclusion, andreverse-phase high-pressure liquid chromatography. The yields ofginsentides from different plant tissues vary >10 fold, with the highestyield from seeds and flowers (0.2-0.4%) and the lowest from roots andleaves (0.01-0.03%). Thus, from 1 kg of dried flowers or seeds nearly400 mg of ginsentides could be obtained.

Table 1 illustrates the amino acid sequence of ginsentides.

FIGS. 18 and 19 shows that ginsentide TP1, TP3 and TP8 relaxesphenylephrine-induced aortic ring contraction, but not KCl-inducedcontracted aortic ring. To examine the vasomotor response of ginsentideTP1, isolated thoracic aorta was pre-contracted with KCl (30 mM) or aselective α1-adrenergic receptor agonist, phenylephrine (0.1 μM).Isometric tension was recorded as a measurement for aortic ringcontraction. The ex-vivo results showed that ginsentide TP1 inhibitsphenylephrine-induced aortic ring contraction in a dose-dependent manner(p<0.05), but not in the KCl-treated group.

Table 5 shows that ginsentide TP1 is metabolically stable

FIG. 20 shows the slow exchange of D2O indicating that the extensivehydrogen bonding network in ginsentide TP1 to confer their structurallystability.

FIG. 21 shows the sequence of TP1, its secondary structure, the overalllay of synthetic and natural TP1, the CPK model of the hydrophobicsurface of TP1 and the 3D structure of TP.

TABLE 5 Ginsentide TP1 is metabolically stable Conditions Initialconcentration remaining (%) Heat (100° C., 2 h) >71 Acid (0.2M HCl, 1h) >99 Trypsin (37° C., 1 h) >87 Chymotrypsin (37° C., 1 h) >90 Pepsin(37° C., 1 h) >99 Human Serum (37° C., 48 h) >92

Example 3. Ginsentides as Nitric Oxide-Boosting Agents

The present invention shows that ginsentide TP1 induces nitric oxidesynthesis. In comparison to the major members of ginsenosides such asRg1 and Rb1 in ginseng, ginsentide TP1 is more effective thanginsenoside Rg1 and Rb1 in nitric oxide synthesis.

Plant peptides and proteins are often underexplored as bioactiveconstituents in medicinal plants. Unlike other peptides and proteins,multiple intramolecular disulfide bonds stabilize cysteine-rich peptidesmaking them extremely stable against heat, acid and enzymaticdegradation. The present invention shows that ginsentide TP1, acysteine-rich peptide, has eight cysteine residues (four stabledisulfide bonds) forming a unique pseudo-cyclic structure, making itresistant to heat, acid, serum proteolytic degradation.

Ginsentides are cysteine-rich peptides and highly cross-linked byintramolecular disulfide bridges. Because ginsentides are 31 amino acidsin length and contain four disulfide bridges, ginsentides have aninside-out arrangement with the inside core filled by the four disulfidelinkages. Consequently, such a structural arrangement forces the bulkyhydrophobic side chains to expose outward, making the surface of themolecule more lipophilic. Indeed, the surface of ginsentide is generallyconsisted of hydrophobic amino acids as determined by 3D NMR. Also,ginsentides are highly compact with an extensive network of of stableintra hydrogen bondings as indicated by the slow exchange of D2O in anNMR study. Intra-hydrogen bondings not only stabilize the ginsentidestructures but also desolvate ginsentides from external hydrogen-bondingwith water. Together, these unique features of ginsentides improve theircell permeability and intestinal absorption, making them bioavailable.In other words, ginsentides have a small-molecule-like stability andoral bioavailability, but a large foot print to increase their on-targetspecificity and decrease their probability of off-target side effectsthan small molecules.

Technical Description of the Invention

Table 1 illustrates the amino acid sequence of ginsentides.

FIG. 22 shows the profiles of ginsentides in the three most commonlyused ginseng species: P. ginseng, P. quinquefolius and P. notoginseng.

FIG. 23 shows the tissue distribution of ginsentides in P. ginseng.

FIG. 24 shows the de novo sequencing of ginsentide TP1.

FIG. 25 shows the disulfide connectivity of ginsentide TP1.

FIG. 26 illustrates the ginsentide-encoding transcripts in all threecommonly used ginseng species: P. ginseng, P. quinquefolius and P.notoginseng.

FIG. 27 shows that ginsentide TP1 increases cellular NO synthesis fromhuman endothelial cells (HUVEC-CS). Comparatively, ginsentide TP1 showsa three-fold increase in potency and magnitude NO release than bothginsenosides Rb1 and Rg1. Ginsentide TP1, TP3, TP8 and synthetic TP1shows comparable cellular NO release.

FIG. 28 shows that ginsentide TP1 induces the accumulation ofphosphorylated eNOS (p-eNOS). 1 μM of ginsentide TP1 induced theaccumulation of p-eNOS in approximately 30 min.

FIG. 29 shows that ginsentide TP1 induced nitric oxide formationinvolves PI3K/Akt signaling.

Table 5 shows that ginsentide TP1 is metabolically stable

FIG. 30 shows the slow exchange of D2O indicating that the extensivehydrogen bonding network in ginsentide TP1 to confer their structurallystability.

FIG. 31 shows that ginsentide TP1 is not cytotoxic to cells andhemolytic to red blood cells. Ginsnetide TP1 is not immunogenic.

Example 4. Ginsentides as Anti-Thrombotic Agents

The present invention shows that ginsentide TP1 has antagonistic effectsagainst P2y12 activation. In comparison to the main ginsenosides inginseng, ginsentide TP1 is more effective than ginsenoside Rg1 and Rb1in antagonizing P2y12 activation. Ginsentide TP1 also exertsanti-platelet aggregative effects in vivo.

Plant peptides and proteins are often underexplored as bioactiveconstituents in medicinal plants. Unlike other peptides and proteins,multiple intramolecular disulfide bonds stabilize cysteine-rich peptidesmaking them extremely stable against heat, acid and enzymaticdegradation. The present invention shows that ginsentide TP1, acysteine-rich peptide, has eight cysteine residues (four stabledisulfide bonds) and a unique pseudo-cyclic structure, making it stableagainst heat, acid, serum and proteolytic degradation.

Cysteine-rich peptides are highly cross-linked by intramoleculardisulfide bridges. This forces the bulky hydrophobic side chains toexpose outward, making the surface of the molecule more lipophilic.Also, the slow exchange of D2O indicates that the hydrogen bonding ofginsentides are intramolecularly connected instead of exposing outwards.These characteristics desolvate ginsentides from water. Therefore, theunique features of ginsentides could improve the intrinsic cellpermeability and intestinal absorption, making it orally active.

Technical Description of the Invention

Table 1 illustrates the amino acid sequence of ginsentides.

FIG. 32 show that ginsentide TP1 antagonize P2y12 activation.Comparatively, ginsentide TP1 is more effective than both ginsenosidesRb1 and Rg1.

FIG. 34 show that ginsentide TP1 inhibits platelet aggregation in vivo

Table 4 illustrates that ginsentide TP1 is stable against heat, acid,proteolytic and serum-mediated degradation.

FIG. 33 shows the slow exchange of D2O indicating that the extensivehydrogen bonding network in ginsentide TP1 to confer their structurallystability.

Example 5. Ginsentides as Anti-Atherosclerotic Agents

The present invention shows that ginsentide TP1 is an endothelial-cellselective adhesion molecule binder that antagonizes monocyte adhesionthat can be useful for the management of atherosclerosis. It is fivetimes more potent than known RGD-derived adhesion inhibitor.

Plant peptides and proteins are often underexplored as bioactiveconstituents in medicinal plants. Unlike other peptides and proteins,multiple intramolecular disulfide bonds stabilize cysteine-rich peptidesmaking them extremely stable against heat, acid and enzymaticdegradation. The present invention shows that ginsentide TP1, acysteine-rich peptide, has eight cysteine residues (four stabledisulfide bonds) and a unique pseudo-cyclic structure, making it stableagainst heat, acid, serum and proteolytic degradation.

Cysteine-rich peptides are highly cross-linked by intramoleculardisulfide bridges. This forces the bulky hydrophobic side chains toexpose outward, making the surface of the molecule more lipophilic.Also, the slow exchange of D2O indicates that the hydrogen bonding ofginsentides are intramolecularly connected instead of exposing outwards.These characteristics desolvate ginsentides from water. Therefore, theunique features of ginsentides could improve the intrinsic cellpermeability and intestinal absorption, making it orally active.

The present invention shows that several ginsentide binds toheparin-affinity column. It is useful for the purification ofginsentides from raw materials.

Identified a new heparin-binding sequence derived from ginsentide thatcan be useful as a new affinity tag for recombinant expression

Technical Description of the Invention

Table 1 illustrates the amino acid sequence of ginsentides.

FIG. 35 shows that ginsentide TP1 shows protein-protein interactionswith endothelial cell selective adhesion molecules (ESAM). ESAM isinvolved in the interaction between monocytes and endothelium.

FIG. 36 shows that ginsentide TP1 antagonists monocyte adhesion and isfive times more potent than known adhesion inhibitor

FIG. 37 shows that several ginsentides are heparin binder and Heparinaffinity chromatography can be used for affinity purification

FIG. 38 shows the identification of a novel heparin binding sequencefrom ginsentide TP1. For recombinant expression of proteins, this newsequence can be inserted at the N-terminal or C-terminal to aid heparinaffinity purification. For facililating efficient cellular binding,heparin binding motifs are known to bind to heparan sulfate proteoglycanand form a major part of the extracellular matrix of cells. This newheparin binding motif can be conjugated to other materials to promotetheir attachment to cell surface. Therefore, this novel heparin bindingsequence could be useful in peptide design and engineering to facilitateefficient cellular binding, as well as being a tag for affinitypurification in recombinant expression

Example 6. Ginsentides as Protective Agents against Doxorubicin-InducedCardiotoxicity

Currently, four strategies have been applied to prevent the occurrenceof DOX-induced cardiotoxicity. But these strategies still haveunavoidable drawbacks.

The first strategy is to adjust the administration schedules of DOX.Some studies showed that administration of DOX via prolonged infusioncan decrease the cardiotoxicity comparing with bolus administration(LEGHA, S. S., et al. Annals of Internal Medicine, 1982. 96(2): p.133-139.). Nevertheless, a randomized trial has shown that infusionadministration preferentially increases the tendency of developingmetastasis in patients (Casper, E. S., et al. Cancer, 1991. 68(6): p.1221-1229.).

The second approach is to modify the structure of DOX and synthesizeanalogues. The most common used structural analogs, epirubicin andmitoxantrone, have less cardiotoxicity than DOX, but theirchemotherapeutic efficacy are lower than DOX (Lehenbauer Ludke, A .R.,et al. Canadian journal of physiology and pharmacology, 2009. 87(10): p.756-763.)

The third strategy is to develop the drug formulation of DOX to controlthe peak concentration after administration. Liposomal encapsulation ofDOX can extends half-life of DOX and increases accumulation in tumortissues rat rather than heart owing to its small size, but there stillexists a possibility of cardiac dysfunction at high accumulative dosage(Abraham, S. A., et al. Methods in enzymology, 2005. 391: p. 71-97.).

The last scheme to reduce the risk of DOX-induced cardiotoxicity iscombination with cardioprotective agents or compounds. However, theyeither interfere with therapeutic potentials of DOX or may inducesecondary malignancies in cancer patients, including dexrazoxane (Tebbi,C. K., et al. Journal of Clinical Oncology, 2007. 25(5): p. 493-500),N-acetylcysteine (Myers, C., et al. Semin Oncol, 1983. 10(Suppl 1): p.53-55), GSH, co-enzyme Q10, vitamin A and α-tocopherol (Legha, S. S., etal. Annals of the New York Academy of Sciences, 1982. 393(1): p.411-417.).

Herein, our preliminary results show that ginsentides attenuatedDOX-induced cardiotoxicity both in zebrafish model and ratcardiomyocytes. Also, ginsentides have no interference with anticancercapacity of DOX during combinational treatment on breast cancer cells.

Ginsentides, a novel class of cysteine-rich-peptides, are highlyresistant to heat, acidic and enzymatic degradation, since they contain31-33 amino acids with 8 cysteine residues and resemble a pseudo-cycliccysteine-knot structure. In addition, pharmacokinetic profiles ofginsentides in mice have shown that they are non-toxic, orallybioavailable and relatively stable in blood. These advantages enhanceginsentides as a highly promising adjuvant cardioprotective agent duringchemotherapy.

Technical Description of the Invention The invention provides anapproach to prevent DOX-induced cardiotoxicity by a combinationaladministration of DOX with ginsentides.

(1) Cardioprotection of Ginsentides in Zebrafish

In this invention, a zebrafish transgenic line Tg(cmIc2:gCaMP) (Chi, N.C., et al. PLoS Biol, 2008. 6(5): p. e109.), which expresses acalcium-sensitive green fluorescent protein (GFP) in cardiomyocytes wasutilized to visualize and evaluate cardiac functions of zebrafishembryos.

No adverse effect of ginsentides in zebrafish was found, even applied athigh concentrations. The major side effects of DOX-inducedcardiotoxicity on zebrafish embryos include (1) reduction of heartbeats,(2) ventricular contractility, and (3) triggering pericardial edema.Addition of ginsentides into DOX-treated zebrafish embryos restored theheartbeat rate near to normal level, and reduced the size of pericardialedema (FIGS. 39 and 40).

To evaluate cardiac functions in zebrafish, ejection fraction (EF) weredetermined by measurement of ventricular volume at the end of systolesand diastoles. EF were markedly reduced after DOX-treatment, but wassignificantly increased and almost returned to normal aftercombinational treatment with ginsentides. (FIG. 41), demonstratingsignificant cardioprotection of ginsentides against DOX-inducedcardiotoxicity in zebrafish.

To identify whether ginsentides can attenuate DOX-induced cardiotoxicityin cardiomyocytes, cell viability after drug treatment was measured byMTT assay. The results showed that ginsentides display no apparenttoxicity in cardiomyoctes even at high concentration, and enhance thegrowth of cardiomyocytes compared to major components in Panax ginseng,ginsenoside Rg1, Rb1 and Re (FIG. 42).

After 24 h co-treatment of DOX with ginsentides, the viability of H9c2cells was enhanced by ˜20% compared to the DOX-alone treatment (FIG.43.). But ginsenoside Rg1, Rb1 and Re did not showed any protectiveeffects on cardiomyoctes during DOX-treatment.

(2) No Interference of Anticancer Effects on Breast Cancer Cells

Next, we studied whether ginsentides would reduce the anticancer effectsof DOX on breast tumor cells. Cell viability of MDA-MB-231 cells was notenhanced after co-treatment of DOX with ginsentides (FIG. 44.).Ginsentides only exerted protective effects on cardiomyocytes and didnot interfere with the therapeutic effects of DOX on tumor cells.

(3) Pharmacokinetics of Ginsentides in Mice

Pharmacokinetic profiles of ginsentides in mice (FIG. 45.) show thatthey are orally bioavailable and relatively stable in blood. Nodiscomfort symptoms were observed in mice after receiving ginsentides,and no death occurred during administration and after a month ofadministration. These advantages provide strong evidence for developingginsentides as a therapeutic agent in clinical use.

Example 7. Ginsentides as Anti-Ageing and Adaptogenic Agents

Most of the anti-ageing agents are commonly grouped as antioxidant,which reduced oxidative stress and protect the cell from unwanted ageingrelated process. In this invention, Ginsentide TP1 is disclosed to be ananti-ageing agent, which directly prevents telomere shortening throughreducing the level of telomeric repeat-binding factor 2 (TERF2).

Unlike antioxidant anti-ageing compounds, ginsentide TP1 not onlyprevents oxidative stress induced ageing but also slow down overallageing process through blocking the telomere shortening process andpromote adaptation to stress.

Unlike conventional peptide drugs, ginsentide TP1 has been proven to bea potential orally active agent due to their excellent stability in bothGI tract as well as serum.

Technical Description of the Invention

To understand the function of ginsentide TP1 at molecular level,HUVEC-CS cells were treated with 1 μM TP1 for a 30 min period. Aftertermination of the treatment, cells were collected through scraping andwashed with ice cold PBS. Cells were lysed in urea lysis buffer (8 Murea in 25 mM TEAB supplemented with protease inhibitor cocktail).Digest 200 μg of protein each using in-solution trypsin digestiontechnique. Resultant tryptic peptides were labeled with TMT reagents,separated by reverse phase fractionation at high pH (pH ˜8) and analyzedusing nano-HPLC coupled Q-Exactive mass spectrometer. Proteinidentification and quantification were performed using mascot searchengine (version 2.4.1) using UniProtKB human protein database and weidentified a total of 5058 proteins with a list of peptides and 1% FDRcutoff. Control condition was used as denominator for TMT based relativeprotein quantitation. Cutoff values of 0.85 and 1.17 were used as thecutoff level of protein abundance change (FIG. 46a ). Using thisapproach, 62 and 113 proteins were identified as up- and down-regulatedin the ginsentide-treated cells (FIG. 46b ). The regulated proteins wereclassified according to their biological functions (FIG. 46c ).

Interestingly the level of telomeric repeat-binding factor 2 (TERF2) wassignificantly decline after 30 min TP1 treatment (FIG. 46d ). Thereduction of TERF2 by TP1 should slow down the telomere shorteningprocess and prevent cellular ageing. Therefore, ginsentide TP1 preventsthe ageing process specifically in vascular cells and maintains thevascular flexibility. It will also reduce other aging related problems.

FIG. 47 shows the effects of ginsentide TP1 on hypoxic endothelial cellproteome. Ginsentide TP1 induced unfolded protein response pathways inhypoxic endothelial cells. It also reduces apoptosis signaling pathwaysand reduced cell adhesion pathways.

FIG. 48-53 shows that ginsentide TP1 promotes adapatation ofendothelials cells to hypoxic stress.

FIG. 54. Ginsentides reduced unfolded proteins in cardiomyocyte,vascular smooth muscle cells, and myocytes.

FIG. 55. Ginsentides reduced reactive oxygen species production inhypoxic endothelial cells.

FIG. 56. Ginsentides increased nitric oxide production in hypoxicendothelial cells.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein. Other embodimentsare within the following claims. In addition, where features or aspectsof the invention are described in terms of Markush groups, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. Further, itwill be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention. Thecompositions, methods, procedures, treatments, molecules and specificcompounds described herein are presently representative of preferredembodiments are exemplary and are not intended as limitations on thescope of the invention. Changes therein and other uses will occur tothose skilled in the art which are encompassed within the spirit of theinvention are defined by the scope of the claims. The listing ordiscussion of a previously published document in this specificationshould not necessarily be taken as an acknowledgement that the documentis part of the state of the art or is common general knowledge.

The invention illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing“, etc. shall be read expansivelyand without limitation. The word “comprise” or variations such as“comprises” or “comprising” will accordingly be understood to imply theinclusion of a stated integer or groups of integers but not theexclusion of any other integer or group of integers. Additionally, theterms and expressions employed herein have been used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by exemplaryembodiments and optional features, modification and variation of theinventions embodied therein herein disclosed may be resorted to by thoseskilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention.

The content of all documents and patent documents cited herein isincorporated by reference in their entirety.

1. A method of solid-phase peptide synthesis of a ginsentide orginsentide-like peptide or a pharmaceutically acceptable salt thereof,said ginsentide or ginsentide-like peptide having (i) the amino acidsequence set forth in any one of SEQ ID NO:1-64; or (ii) the amino acidsequence sharing at least 65% sequence identity with the peptide of (i)over its entire sequence, wherein the method comprises the steps of: (a)synthesizing the ginsentide or ginsentide-like peptide on a solidsupport by stepwise coupling of Fmoc-protected; (b) cleaving theginsentide or ginsentide-like peptide from the solid support anddeprotecting the peptide; and subsequently, (c) performing organicoxidative folding of the ginsentide or ginsentide-like peptide to formnative disulfide connections.
 2. The method of claim 1, wherein step (a)comprises the steps of: (i) deprotecting a first amino acid linked tothe solid support by removing protective chemical groups from the firstamino acid; (ii) activating chemical groups on a second amino acid toprepare the second amino acid for coupling with the first amino acid;(iii) coupling the activated second amino acid to the deprotected firstamino acid to form a peptide from the first and second amino acids; and(iv) successively deprotecting, and coupling a plurality of amino acidsinto the growing peptide chain until the ginsentide or ginsentide-likepeptide is synthesized.
 3. The method of claim 2, wherein the methodcomprises accelerating at least one of the deprotecting, activating, andcoupling steps by applying microwave energy.
 4. The method of claim 1,wherein the organic oxidative folding is performed in non-aqueousconditions and performed in organic solvents.
 5. The method of claim 1,wherein the organic oxidative folding comprises folding the ginsentideor ginsentide-like peptide in an organic solvent comprising cysteamineand/or morpholine.
 6. The method of claim 1, wherein the organicoxidative folding is performed at 4° C.
 7. The method of claim 1,wherein the method comprises purifying the folded ginsentide orginsentide-like peptide or salt thereof.
 8. A method of recombinantlypreparing a ginsentide or ginsentide-like peptide or a pharmaceuticallyacceptable salt thereof, said ginsentide or ginsentide-like peptidehaving (i) the amino acid sequence set forth in any one of SEQ IDNO:1-64; or (ii) the amino acid sequence sharing at least 65% sequenceidentity with the peptide of (i) over its entire sequence, wherein themethod comprises the steps of: (a) providing a host cell comprising apolynucleotide, wherein the polynucleotide encodes a polypeptidecomprising Maltose-binding protein having the amino acid sequence setforth in SEQ ID NO:65, a enterokinase cleavage sequence having the aminoacid sequence set forth in SEQ ID NO:66, and the ginsentide orginsentide-like peptide or salt thereof; (b) culturing the host cell ina growth medium under conditions allowing production of the polypeptide,and recovering the polypeptide from the medium; and (c) generating theginsentide or ginsentide-like peptide or salt thereof by cleaving thepolypeptide using enterokinase.
 9. The method of claim 8, wherein thepolypeptide has the amino acid sequence set forth in any one of SEQ IDNOs: 67-80.
 10. The method of claim 8, wherein the method furthercomprises purification of the recombinantly produced polypeptide priorto the enterokinase cleavage.
 11. The method of claim 8, wherein themethod further comprises purification of the ginsentide orginsentide-like peptide or salt thereof by anion-exchange chromatographyand/or reverse-phase chromatography after the enterokinase cleavage. 12.A pharmaceutical composition comprising a ginsentide or ginsentide-likepeptide or a pharmaceutically acceptable salt, said ginsentide orginsentide-like peptide having (i) the amino acid sequence set forth inany one of SEQ ID NO:1-64; or (ii) the amino acid sequence sharing atleast 65% sequence identity with the peptide of (i) over its entiresequence.
 13. A method of treating and/or preventing thrombosis in asubject in need thereof, the method comprising administering to thesubject the pharmaceutical composition of claim 12.